CN111868255A - Methods and reagents for enriching nucleic acid material for sequencing applications and other nucleic acid material interrogation - Google Patents

Abstract

The present technology generally relates to methods and compositions for targeted nucleic acid sequence enrichment, and the use of such enrichment for error-corrected nucleic acid sequencing applications and other nucleic acid sequence interrogation. In some embodiments, the provided methods provide non-amplification targeted based enrichment strategies compatible with the use of molecular barcodes for error correction. Other embodiments provide methods for non-amplification targeted enrichment strategies that are compatible with direct digital sequencing (DDS) and other sequencing strategies that do not use molecular barcodes (eg, single-molecule sequencing modes and interrogation).

Description

Methods and reagents for enriching nucleic acid material for sequencing applications and other nucleic acid material interrogation

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of US Provisional Patent Application No. 62/643,738, filed March 15, 2018, the disclosure of which is incorporated herein by reference in its entirety.

Background technique

Various approaches have been developed at the protocol development, chemical/biochemical, and data processing levels to mitigate the effects of PCR-based errors in massively parallel sequencing (MPS, sometimes referred to as next-generation DNA sequencing, NGS) applications. Furthermore, before or during amplification, PCR replicates from individual DNA fragments can be based on unique random splice sites or by exogenous labeling (i.e., using molecular barcodes, also known as molecular tags, unique molecular The techniques in which identifiers [UMI] and single molecule identifiers [SMI]) are resolved are also commonly used. This method has been used to improve the counting accuracy of DNA and RNA templates. Because all amplicons from a single starting molecule can be unambiguously identified, any changes in the sequence of identically labeled sequencing reads can be used to correct for base errors that occur during PCR or sequencing. For example, Kinde et al. (ProcNatl Acad SciUSA 108, 9530-9535, 2011) introduced SafeSeqS, which uses single-stranded molecular barcodes, to reduce the error rate of sequencing by grouping and concordantly sequenced PCR copies that share barcodes. However, the introduction of single-stranded molecular barcodes did not completely eliminate the PCR artifact that appeared in the first round of amplification, which was carried over to the derived copies as a "jackpot" event.

Methods for higher precision genotyping of single nucleotide polymorphism (SNP) loci, short tandem repeat (STR) loci and many other forms of mutations and genetic variants in medicine, forensics, genotoxicity Various applications in science and other scientific industrial applications are desired. One challenge, however, is how to most efficiently generate sequence information from as many copies of related genetic material that are sequenced as possible, with the highest confidence but at a reasonable cost. Various consistent sequencing methods (both molecular barcode-based and non-molecular barcode-based) have been successfully used for error correction to help better identify variants in mixtures (see J. Salk et al, Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations, Nature ReviewsGenetics, 2018, for detailed discussion), but there are various tradeoffs in performance. We have previously described double-stranded sequencing, an ultra-high-precision sequencing method that relies on genotyping and comparing the sequences of independent strands of double-stranded nucleic acid molecules for error correction purposes. Aspects of the techniques set forth herein describe methods for improving cost efficiency, recovery efficiency, and other performance metrics, as well as overall processing speed for double-stranded sequencing and other sequencing applications, for achieving high precision sequencing reads.

SUMMARY OF THE INVENTION

The present technology generally relates to methods for targeted nucleic acid sequence enrichment and the use of such enrichment for error-corrected nucleic acid sequencing applications and other nucleic acid material interrogation. In some embodiments, highly accurate, error-corrected, and massively parallel sequencing of nucleic acid material is possible using target nucleic acid material that has been enriched from a sample. In some aspects, the target-enriched nucleic acid material is double-stranded, and the one or more methods of uniquely labeling strands of a double-stranded nucleic acid complex can be used in such a way that each strand can be informationally linked to its complementary strand. However, each strand or amplification product derived therefrom can also be distinguished after sequencing it, and this information can be further used for the purpose of error correction of the determined sequences. Some aspects of the present technology provide methods and compositions for improving cost, conversion rate of sequenced molecules, and time efficiency of generating labeled molecules for targeted ultra-high precision sequencing. In some embodiments, the provided methods and compositions allow for accurate analysis of very small amounts of nucleic acid material (eg, DNA from small clinical samples or freely floating in blood or samples taken from crime scenes). In some embodiments, the provided methods and compositions allow detection of nucleic acid material in samples of less than one percent of cells or molecules (eg, less than one in one thousand cells or molecules, less than one in ten thousand). A mutation that exists at a frequency of one cell or molecule, less than one in 100,000 cells or molecules).

Aspects of the present technology relate to methods for enriching target nucleic acid material, the method comprising providing the nucleic acid material, and cleaving the nucleic acid material with one or more targeted endonucleases such that a target region of a predetermined length is bound to the nucleic acid material the rest of the separation. The method may further comprise enzymatically destroying the non-targeted nucleic acid material, releasing the target region of predetermined length from the targeted endonuclease; and analyzing the cleaved target region.

Additional aspects of the present technology relate to methods for enriching target nucleic acid material, the method comprising providing the nucleic acid material, cleaving the nucleic acid material with one or more targeted endonucleases such that a target region of predetermined length is bound to the nucleic acid The remainder of the material is isolated, wherein the at least one targeted endonuclease includes a capture label; a target region of a predetermined length is captured with an extraction moiety configured to bind the capture label; the predetermined length is released from the targeted endonuclease the target region; and the target region for analysis of cleavage.

Additional aspects of the present technology relate to methods for enriching a target nucleic acid material, comprising providing the nucleic acid material; binding a catalytically inactive CRISPR-associated (Cas) enzyme to a target region of the nucleic acid material; digesting with one or more nucleic acids Enzymatic treatment of nucleic acid material by enzymes such that non-targeted nucleic acid material is destroyed and the target region is protected from digestive enzymes by the bound catalytically inactive Cas enzyme; the target region is released from the catalytically inactive Cas enzyme ; and analyzing target regions.

Another aspect of the present technology relates to a method for enriching a target nucleic acid material, comprising providing a nucleic acid material; providing a pair of catalytically active targeted endonucleases and at least one catalytically inactive targeted endonuclease comprising a capture label An endonuclease, wherein the catalytically inactive targeted endonuclease is directed to bind to a target region of a nucleic acid material, and wherein the catalytically active targeted endonuclease is directed to bind to a catalytically inactive the target region on either side of the targeted endonuclease; cleave the nucleic acid material with the catalytically active targeted endonuclease such that the target region is separated from the rest of the nucleic acid material; with the pair configured to bind The extraction portion of the capture label captures the target region; releases the target region from the targeted endonuclease; and analyzes the cleaved target region.

A further aspect includes a method for enriching a target nucleic acid material from a sample comprising a plurality of nucleic acid fragments, comprising providing the sample comprising the target nucleic acid fragments and non-target nucleic acid fragments with one or more catalytically inactive catalysts having capture labels. CRISPR-associated (Cas) enzymes, wherein the one or more catalytically inactive Cas enzymes are configured to bind target nucleic acid fragments; providing a surface comprising an extraction portion configured to bind a capture label; and by The target nucleic acid fragments are captured via the extraction moiety in conjunction with the capture label, and the target nucleic acid fragments are separated from the non-target nucleic acid fragments.

Various embodiments provide methods for enriching a target double-stranded nucleic acid material, comprising providing the nucleic acid material; cleaving the nucleic acid material with one or more targeted endonucleases to generate double-stranded target nucleic acid fragments, the double-stranded target nucleic acid fragments. The strand target nucleic acid fragment includes a 5' cohesive end having a 5' predetermined nucleotide sequence and/or a 3' cohesive end having a 3' predetermined nucleotide sequence; and passes through at least one of the 5' cohesive end and the 3' cohesive end The double-stranded target nucleic acid molecule is separated from the remainder of the nucleic acid material.

Additional embodiments provide kits for enriching target nucleic acid material comprising a nucleic acid library comprising nucleic acid material and a plurality of catalytically inactive Cas enzymes, wherein the Cas enzymes comprise tags with sequence codes , and wherein the plurality of Cas enzymes bind to a plurality of site-specific target regions along the nucleic acid material. The kit further includes a plurality of probes, wherein each probe includes an oligonucleotide sequence including the complement of the corresponding sequence code and a capture label. The kit may also include a look-up table that classifies the relationship between the site-specific target regions, the sequence codes associated with the site-specific target regions, and probes that include the complement of the corresponding sequence codes.

In some embodiments, error-corrected sequence reads are used to identify or characterize cancer, cancer risk, cancer mutation, cancer metabolic state, mutant phenotype, oncogenicity in the organism or subject from which the double-stranded target nucleic acid molecule is derived drug exposure, toxin exposure, chronic inflammatory exposure, age, neurodegenerative diseases, pathogens, drug-resistant variants, fetal molecules, forensically relevant molecules, immunologically relevant molecules, mutated T cell receptors, mutated B cell receptors, Mutated immunoglobulin loci, kategis sites in the genome, hypermutation sites in the genome, low frequency variants, subclonal variants, minority molecular populations, sources of contamination, nucleic acid synthesis errors, enzymatic modification errors, chemical modification errors , gene editing errors, gene therapy errors, nucleic acid information storage fragments, microbial quasispecies, viral quasispecies, organ transplantation, organ transplant rejection, cancer recurrence, residual cancer after treatment, pre-neoplastic state, dysplasia state, microchimerism, A stem cell transplant state, a cell therapy state, a nucleic acid marker attached to another molecule, or a combination thereof. In some embodiments, error-corrected sequence reads are used to identify oncogenic compounds or exposures. In some embodiments, error-corrected sequence reads are used to identify mutagenic compounds or exposures. In some embodiments, the nucleic acid material is derived from a forensic sample and the error-corrected sequence reads are used for forensic analysis.

In some embodiments, the single-molecule identifier sequence includes an endogenous cleavage point or an endogenous sequence that can be associated with a cleavage point location. In some embodiments, the single-molecule identifier sequence is at least one of a degenerate or semi-degenerate barcode sequence, one or more nucleic acid fragment ends of nucleic acid material, or a combination thereof, which uniquely labels double-stranded nucleic acid molecules. In some embodiments, the adaptor and/or adaptor sequence includes at least one nucleotide position that is at least partially non-complementary or includes at least one non-standard base. In some embodiments, the adaptor comprises a single "U-shaped" oligonucleotide sequence formed from about 5 or more self-complementary nucleotides.

According to various embodiments, any of a variety of nucleic acid materials may be used. In some embodiments, the nucleic acid material can include at least one modification to a polynucleotide within a typical sugar-phosphate backbone. In some embodiments, the nucleic acid material can include at least one modification within any base in the nucleic acid material. For example, by way of non-limiting example, in some embodiments, the nucleic acid material is or includes at least one of double-stranded DNA, double-stranded RNA, peptide nucleic acid (PAN), locked nucleic acid (LNA).

In some embodiments, the provided methods further comprise attaching an adaptor molecule to the double-stranded nucleic acid molecule. In some embodiments, the linking step comprises linking the double-stranded nucleic acid material to at least one double-stranded degenerate barcode sequence to form a double-stranded nucleic acid molecule barcode complex, wherein the double-stranded degenerate barcode sequence is in each strand Include single molecule identifier sequences. In some embodiments, the double-stranded nucleic acid molecule is a double-stranded DNA molecule or a double-stranded RNA molecule. In some embodiments, the double-stranded nucleic acid molecule includes at least one modified nucleotide or non-nucleotide molecule.

In some embodiments, the ligation includes the activity of at least one ligase. In some embodiments, the at least one ligase is selected from the group consisting of DNA ligases and RNA ligases. In some embodiments, the ligation comprises ligase activity at the ligation domain associated with the adaptor molecule. In some embodiments, the ligation comprises ligase activity at the ligation domain associated with the adaptor molecule and the ligable end of the nucleic acid molecule. In some embodiments, the linking domains and linkable ends of the double-stranded nucleic acid molecule are compatible (eg, have single-stranded regions that are complementary to each other). In some embodiments, the linking domain is a nucleotide sequence from or related to one or more degenerate or semi-degenerate nucleotides. In some embodiments, the linking domain is a nucleotide sequence from one or more non-degenerate nucleotides. In some embodiments, the linking domain contains one or more modified nucleotides. In some embodiments, the ligation domain and/or ligatable ends include T-overhangs, A-overhangs, CG-overhangs, blunt ends, recombination sequences, endonuclease cleavage site overhangs, restriction digests overhangs or another linkable region. In some embodiments, at least one chain of the linking domain is phosphorylated. In some embodiments, the linking domain includes an endonuclease cleavage sequence or a portion thereof.

In some embodiments, endonuclease cleavage sequences are cleaved by endonucleases (eg, tunable endonucleases, restriction endonucleases) to produce blunt ends or overhangs with ligable regions. In some embodiments, the ligable end of the double-stranded nucleic acid molecule includes an endonuclease cleavage sequence or a portion thereof. In some embodiments, endonuclease (eg, programmable/targeted endonuclease, restriction endonuclease) production comprises having a known nucleotide length (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) and "sticky ends" of the sequence or The overhang of the single-stranded overhang region.

In some embodiments, the identifier sequence is or includes a single molecule identifier (SMI) sequence. In some embodiments, the SMI sequence is an endogenous SMI sequence. In some embodiments, the endogenous SMI sequence is associated with a splice point. In some embodiments, the SMI sequence includes at least one degenerate or semi-degenerate nucleic acid. In some embodiments, the SMI sequence is non-degenerate. In some embodiments, the SMI sequence is a nucleotide sequence of one or more degenerate or semi-degenerate nucleotides. In some embodiments, the SMI sequence is a nucleotide sequence of one or more non-degenerate nucleotides. In some embodiments, the SMI sequence includes at least one modified nucleotide or non-nucleotide molecule. In some embodiments, the SMI sequence includes a primer binding domain.

Super

锁核酸、5-硝基吲哚、2'-O-甲基RNA碱基、羟甲基dC、异dG、异-dC、氟代C、氟代U、氟代A、氟代G、2-甲氧基乙氧基A、2-甲氧基乙氧基MeC、2-甲氧基乙氧基G、2-甲氧基乙氧基T、8-氧代-A、8-氧代G、5-羟甲基-2'-脱氧胞苷、5'-甲基异胞嘧啶、四氢呋喃、异胞嘧啶、异鸟苷、尿嘧啶、甲基化核苷酸、RNA核苷酸、核糖核苷酸、8-氧代-G、BrdU、Loto dU、呋喃、荧光染料、叠氮化物核苷酸、脱碱基核苷酸、5-硝基吲哚核苷酸和洋地黄毒苷核苷酸。In some embodiments, the modified nucleotide or non-nucleotide molecule is selected from the group consisting of 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, trans dT, trans-dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinosine, Super

Super

Locked Nucleic Acid, 5-Nitroindole, 2'-O-Methyl RNA Base, Hydroxymethyl dC, Iso-dG, Iso-dC, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G, 2 -Methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, 2-methoxyethoxy T, 8-oxo-A, 8-oxo G, 5-hydroxymethyl-2'-deoxycytidine, 5'-methylisocytosine, tetrahydrofuran, isocytosine, isoguanosine, uracil, methylated nucleotides, RNA nucleotides, ribose Nucleotides, 8-oxo-G, BrdU, Loto dU, furans, fluorescent dyes, azide nucleotides, abasic nucleotides, 5-nitroindole nucleotides, and digoxigenin nucleosides acid.

In some embodiments, the cleavage site is or includes a restriction endonuclease recognition sequence. In some embodiments, the cleavage site is or includes a user-directed recognition sequence for a targeted endonuclease (eg, CRISPR or CRISPR-like endonuclease) or other tunable endonuclease. In some embodiments, cleavage of the nucleic acid material can include at least one of: enzymatic digestion, enzymatic cleavage, enzymatic cleavage of one strand, enzymatic cleavage of both strands, incorporation of modified nucleic acid followed by enzymatic treatment (which results in a or cleavage of both strands), incorporation of replication blocking nucleotides, incorporation of chain terminators, incorporation of photocleavable linkers, incorporation of uracil, incorporation of ribobases, incorporation of 8-oxo-ornithine Purine adducts, use of restriction endonucleases, use of ribonucleoprotein endonucleases (e.g. Cas enzymes such as Cas9 or CPF1) or other programmable endonucleases (e.g. homing endonucleases, zinc fingers Nucleases, TALENs, meganucleases (eg, megaTAL nucleases, arginine nucleases, etc.), and any combination thereof.

In some embodiments, the capture label is or includes at least one of the following: acridine, azide, azide (NHS ester), digoxigenin (NHS ester), I-linker, amino modifier C6, amino modifier C12, amino modifier C6dT, Unilink amino modifier, hexynyl, 5-octadiynyl dU, biotin, biotin (azide), biotin dT, biotin TEG , Bibiotin, PC Biotin, Dethiobiotin TEG, Thiol Modifier C3, Dithiol, Thiol Modifier C6 S-S and Succinyl Groups.

In some embodiments, the extraction moiety is or includes aminosilane, epoxysilane, isothiocyanate, aminophenylsilane, aminopropylsilane, mercaptosilane, aldehyde, epoxide, phosphonate, streptavidin At least one of and at least one of avidin, avidin, a hapten that recognizes an antibody, a specific nucleic acid sequence, magnetically attractive particles (Dynabeads), and a photolabile resin.

In some embodiments, the provided methods further comprise amplifying the nucleic acid material by using primers specific for the adaptor sequence and/or by using primers specific for the non-adapter portion of the nucleic acid product. It is contemplated that any of a variety of methods for amplifying nucleic acid material may be used in accordance with various embodiments. For example, in some embodiments, at least one amplification step comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), isothermal amplification, polymerase clonal amplification in emulsion Amplification, bridging amplification on the surface, on the surface of a bead, or within a hydrogel, and any combination thereof. In some embodiments, amplifying the nucleic acid material comprises using regions that are at least partially complementary to the first adaptor sequence and the second adaptor sequence (eg, with regions on the 5' and/or 3' ends of each strand of the nucleic acid material. single-stranded oligonucleotides that are at least partially complementary to the adaptor sequences). In some embodiments, amplifying the nucleic acid material comprises using single-stranded oligonucleotides that are at least partially complementary to regions of the relevant genomic sequence and single-stranded oligonucleotides that are at least partially complementary to regions of adaptor sequences.

In some embodiments, amplifying the nucleic acid material comprises generating a plurality of amplicons derived from the first strand and a plurality of amplicons derived from the second strand.

In some embodiments, the provided methods further comprise the steps of cleaving the nucleic acid material with one or more targeted endonucleases such that target nucleic acid fragments of substantially known length are formed, and length to separate target nucleic acid fragments. In some embodiments, provided methods further comprise ligating an adaptor (eg, an adaptor sequence) to a target nucleic acid (eg, a target nucleic acid fragment) of substantially known length (eg, after a size enrichment step).

In some embodiments, the nucleic acid material can be or include one or more target nucleic acid fragments. In some embodiments, the one or more target nucleic acid fragments each comprise related genomic sequences from one or more locations in the genome. In some embodiments, the one or more target nucleic acid fragments comprise targeted sequences from substantially known regions of the nucleic acid material. In some embodiments, isolating target nucleic acid fragments based on substantially known lengths comprises enriching target nucleic acid fragments by gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, filtration, or SPRI bead purification.

In some embodiments, the provided methods further comprise the step of cleaving the double-stranded nucleic acid material with one or more targeted endonucleases such that double-stranded target nucleic acid fragments are formed, the double-stranded target nucleic acid fragments comprising One or both ends of a sequence having substantially known lengths and/or single-stranded overhangs. In some embodiments, provided methods further comprise the step of isolating double-stranded target nucleic acid fragments based on substantially known lengths and/or sequences of single-stranded overhangs. In some embodiments, provided methods further comprise ligating an adaptor (eg, an adaptor sequence) to a double-stranded target nucleic acid (eg, a target nucleic acid fragment) having a substantially known length and/or sequence of single-stranded overhangs . In some embodiments, the double-stranded target nucleic acid can have ligatable ends that are substantially uniquely compatible (eg, complementary) to the ligation domains of the ligation-selected adaptor molecule, such that targets from substantially known regions within the nucleic acid material are included One or more target nucleic acid fragments of a targeted sequence can be selectively enriched by amplification with primers specific for the adaptor sequence associated with the ligated selected adaptor.

According to various embodiments, some of the provided methods can be used to sequence any of various suboptimal (eg, damaged or degraded) samples of nucleic acid material. For example, in some embodiments, at least some of the nucleic acid material is damaged. In some embodiments, the damage is or includes oxidation, alkylation, deamination, methylation, hydrolysis, hydroxylation, nicking, intrachain crosslinks, interchain crosslinks, blunt end chain breaks, staggered end double strand breaks , phosphorylation, dephosphorylation, ubiquitination, glycosylation, deglycosylation, putrescylation, carboxylation, halogenation, formylation, single-strand gap, damage caused by heat, caused by drying damage from UV exposure, damage from gamma radiation, damage from X-radiation, damage from ionizing radiation, damage from non-ionizing radiation, damage from heavy particle radiation, damage from nuclear decay damage caused by beta radiation, damage caused by alpha radiation, damage caused by neutron radiation, damage caused by proton radiation, damage caused by cosmic radiation, damage caused by high pH, damage caused by low pH Damage caused by reactive oxidizing species, damage caused by free radicals, damage caused by peroxides, damage caused by hypochlorite, damage caused by tissue fixation such as formalin or formaldehyde damage, damage caused by active iron, damage caused by low ionic conditions, damage caused by high ionic conditions, damage caused by unbuffered conditions, damage caused by nucleases, damage caused by environmental exposure, caused by fire damage caused by mechanical stress, damage caused by enzymatic degradation, damage caused by microorganisms, damage caused by preparative mechanical shearing, damage caused by preparative enzymatic cleavage, damage naturally occurring in vivo, in Damage occurring during nucleic acid extraction, Damage occurring during sequencing library preparation, Damage introduced by polymerase, Damage introduced during nucleic acid repair, Damage occurring during nucleic acid end tailing, Damage occurring during nucleic acid ligation, Damage occurring during nucleic acid ligation, Damage that occurs during sequencing, damage that occurs due to mechanical processing of DNA, damage that occurs during passage through a nanopore, damage that occurs as part of aging in an organism, damage that occurs due to chemical exposure of an individual, damage that occurs due to induced Damage due to mutagenic agents, damage due to carcinogens, damage due to cleavage agents, damage due to in vivo inflammatory damage due to oxygen exposure, damage due to one or more strand breaks, and their At least one of any combination.

It is contemplated that nucleic acid material can come from a variety of sources. For example, in some embodiments, nucleic acid material (eg, comprising one or more double-stranded nucleic acid molecules) is provided from a sample from a human subject, animal, plant, fungus, virus, bacteria, protozoa, or any other life form ). In other embodiments, the sample includes nucleic acid material that has been at least partially synthesized. In some embodiments, the sample is or includes body tissue, biopsy sample, skin sample, blood, serum, plasma, sweat, saliva, cerebrospinal fluid, mucus, uterine lavage, vaginal swab, Pap smear, nasal swab , oral swabs, tissue scrapings, hair, fingerprints, urine, feces, vitreous humor, peritoneal lavage, sputum, bronchial lavage, oral lavage, pleural lavage, gastric lavage, gastric juice, Bile, pancreatic duct lavage, bile duct lavage, common bile duct lavage, gallbladder fluid, synovial fluid, infected wounds, uninfected wounds, archaeological samples, forensic samples, water samples, tissue samples, food samples, biological Reactor samples, plant samples, bacterial samples, protozoa samples, fungal samples, animal samples, viral samples, multi-organism samples, nail scrapings, semen, prostatic fluid, vaginal fluid, vaginal swabs, tubal lavage fluid, acellular Nucleic acids, intracellular nucleic acids, metagenomic samples, lavages or swabs of implanted foreign bodies, nasal lavages, intestinal fluids, epithelial brushes, epithelial lavages, tissue biopsy samples, autopsy samples, necropsy Samples, organ samples, human identification samples, non-human identification samples, artificially generated nucleic acid samples, synthetic gene samples, banked or stored nucleic acid samples, tumor tissue, fetal samples, organ transplant samples, microbial culture samples, nuclear DNA samples, Mitochondrial DNA samples, chloroplast DNA samples, acroplast DNA samples, organelle samples, and any combination thereof. In some embodiments, the nucleic acid material is from more than one source.

As described herein, in some embodiments, it may be advantageous to process nucleic acid material in order to increase the efficiency, accuracy, and/or speed of the sequencing process. In some embodiments, the nucleic acid material comprises nucleic acid molecules of substantially uniform length and/or of substantially known length. In some embodiments, the substantially uniform length and/or the substantially known length is between about 1 and about 1,000,000 bases. For example, in some embodiments, the substantially uniform length and/or the substantially known length may be at least 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 15; 20; 25 ;30;35;40;50;60;70;80;90;100;120;150;200;300;400;500;600;700;800;900;1000;1200;1500;2000;3000;4000 5000; 6000; 7000; 8000; 9000; 10,000; 15,000; 20,000; 30,000; 40,000; or 50,000 bases in length. In some embodiments, the substantially uniform length and/or the substantially known length may be at most 60,000; 70,000; 80,000; 90,000; 100,000; 120,000; ; 900,000; or 1,000,000 bases. As a specific non-limiting example, in some embodiments, the substantially uniform length and/or the substantially known length is from about 100 to about 500 bases. In some embodiments, the methods described herein include the step of targeting enriched nucleic acid material, thereby providing nucleic acid molecules of one or more lengths and/or of substantially known lengths. In some embodiments, the nucleic acid material is cleaved into nucleic acid molecules of substantially uniform length and/or of substantially known length by one or more targeted endonucleases. In some embodiments, the targeted endonuclease includes at least one modification.

In some embodiments, the nucleic acid material includes nucleic acid molecules of length within one or more substantially known size ranges. In some embodiments, nucleic acid molecules can be 1 to about 1,000,000 bases, about 10 to about 10,000 bases, about 100 to about 1000 bases, about 100 to about 600 bases, about 100 to about 500 bases bases, or some combination of them.

In some embodiments, the targeted endonuclease is or includes a restriction endonuclease (ie, a restriction enzyme) that cleaves DNA at or near the recognition site (eg, EcoRI, BamHI, XbaI, HindIII, At least one of AluI, AvaII, BsaJI, BstNI, DsaV, Fnu4HI, HaeIII, MaeIII, N1aIV, NSil, MspJI, FspEI, NaeI, Bsu36I, NotI, HinF1, Sau3AI, PvuII, SmaI, HgaI, AluI, EcoRV, etc.) . Lists of several restriction endonucleases are available in printed and computer readable form and are provided by a number of commercial suppliers (eg, New England Biolabs, Ipswich, MA). One of ordinary skill in the art will appreciate that any restriction endonuclease may be used in accordance with various embodiments of the present technology. In other embodiments, the targeted endonuclease is or includes at least one of a ribonucleoprotein complex, such as, for example, a CRISPR-associated (Cas) enzyme/guide RNA complex (eg, Cas9 or Cpf1) or a Cas9-like enzymes. In other embodiments, the targeted endonuclease is or includes a homing endonuclease, zinc finger nuclease, TALEN and/or meganuclease (eg, megaTAL nuclease, etc.), arginine nuclease, or its combination. In some embodiments, the targeted endonuclease includes Cas9 or CPF1 or a derivative thereof. In some embodiments, more than one targeted endonuclease may be used (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the targeted endonuclease can be used to cleave more than one potential target region (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the nucleic acid material multiple). In some embodiments, where there is more than one target region of nucleic acid material, each target region may have the same (or substantially the same) length. In some embodiments, where there is more than one target region of nucleic acid material, at least two of the target regions of known length differ in length (eg, the first target region has a length of 100 bp, and the second The two target regions have a length of 1,000 bp).

In some embodiments, at least one amplification step comprises at least one primer and/or adaptor sequence that is or includes at least one non-standard nucleotide. As a further example, in some embodiments, at least one adaptor sequence is or includes at least one non-standard nucleotide. In some embodiments, the non-standard nucleotides are selected from the group consisting of uracil, methylated nucleotides, RNA nucleotides, ribonucleotides, 8-oxo-guanine, biotinylated nucleotides, desulfurized Biotin nucleotides, thiol-modified nucleotides, acrylate-modified nucleotides, iso-dC, iso-dG, 2'-O-methyl nucleotides, inosine nucleotides locked nucleic acids, peptide nucleic acids , 5-methyl dC, 5-bromodeoxyuridine, 2,6-diaminopurine, 2-aminopurine nucleotide, abasic nucleotide, 5-nitroindole nucleotide, adenylated nucleus nucleotides, azide nucleotides, digoxigenin nucleotides, I-linkers, 5'hexynyl modified nucleotides, 5-octadiynyl dU, photocleavable spacers, non-removable Photocleavable spacers, click chemistry compatible modified nucleotides, fluorescent dyes, biotin, furan, BrdU, fluoro-dU, loto-dU, and any combination thereof.

According to several embodiments, any of a variety of analysis steps may be used in order to improve one or more of the accuracy, speed, and efficiency of the provided process. For example, in some embodiments, sequencing each of the first nucleic acid strand and the second nucleic acid strand of the double-stranded nucleic acid molecule comprises comparing the sequences of the multiple strands derived from the first nucleic acid strand to determine the first strand consensus sequence , and comparing the sequences of the multiple strands derived from the second nucleic acid strand to determine the second strand consensus sequence. In some embodiments, comparing the sequence of the first nucleic acid strand to the sequence of the second nucleic acid strand includes comparing the first strand consensus sequence and the second strand consensus sequence to provide an error-corrected consensus sequence. In other embodiments, the error-corrected sequence of a double-stranded target nucleic acid molecule can be determined by comparing a single sequence read from a first nucleic acid strand to a single sequence read from a second nucleic acid strand.

One aspect provided by some embodiments is the ability to generate high quality sequencing information from very small amounts of nucleic acid material. In some embodiments, provided methods and compositions can be combined with up to about 1 picogram (pg); 10 pg; 100 pg; 1 nanogram (ng); 10 ng; 100 ng; , 800ng, 900ng or 1000ng of starting nucleic acid material were used together. In some embodiments, the provided methods and compositions can be used with an input amount of nucleic acid material of up to 1 molecular copy or genomic equivalent, 10 molecular copies or genomic equivalent thereof, 100 Molecular copies or genomic equivalents thereof, 1,000 molecular copies or genomic equivalents thereof, 10,000 molecular copies or genomic equivalents thereof, 100,000 molecular copies or genomic equivalents thereof, or 1,000,000 molecular copies or genomic equivalents thereof. For example, in some embodiments, up to 1,000 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 10 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 pg of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 pg of nucleic acid material is initially provided for a particular sequencing process.

As used in this application, the terms "about" and "approximately" are used as equivalents. Any reference herein to a publication, patent or patent application is incorporated by reference in its entirety. Any use of numbers with or without about/approximate in this application is intended to cover any normal fluctuations as understood by one of ordinary skill in the relevant art.

In various embodiments, the enrichment of nucleic acid material is provided at a faster rate (eg, with fewer steps) and at a lower cost (eg, using fewer reagents), comprising enriching the nucleic acid material to a relevant area and result in an increase in the required data. Various aspects of the present technology have numerous applications in preclinical and clinical testing and diagnostics, as well as other applications.

Specific details of several embodiments of the technology are described below and with reference to Figures 1-22C. Although many of the examples are described herein with respect to double-stranded sequencing, other sequencing modalities capable of generating error-corrected sequencing reads, other sequencing modalities for providing sequence information, in addition to those described herein, are also in the present technology In the range. In addition, other nucleic acid interrogations are expected to benefit from the nucleic acid enrichment methods and reagents described herein. Furthermore, other embodiments of the present technology may have configurations, components, or procedures other than those described herein. Accordingly, those of ordinary skill in the art will accordingly understand that the technology can have other embodiments with additional elements, and that the technology can have other embodiments without several of the features shown and described below with reference to FIGS. 1-22C .

Description of drawings

Many aspects of the present disclosure may be better understood with reference to the following drawings. Components in the figures are not necessarily drawn to scale. Rather, emphasis is placed upon clearly illustrating the principles of the present disclosure.

1 is a graph plotting the relationship between nucleic acid insert size and resulting family size after amplification, according to embodiments of the present technology.

2A and 2B are schematic diagrams illustrating sequencing data generated for different nucleic acid insert sizes in accordance with aspects of the present technology.

3 is a schematic diagram illustrating the steps of a method for generating targeted fragment sizes using CRISPR/Cas9 according to an embodiment of the present technology. Panel A shows gRNA-facilitated binding of Cas9 at the targeted DNA site. Cas9-directed cleavage releases blunt-ended double-stranded target DNA fragments of known length, as shown in panel B. Panel C depicts further processing steps for positive enrichment/selection of target DNA fragments by size selection. Optionally, as depicted in Figure D , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing.

4 is a schematic diagram illustrating the steps of a method for generating targeted nucleic acid fragments of known/selected lengths using CRISPR/Cas9 variants, according to embodiments of the present technology. Using the CRISPR/Cas9 ribonucleoprotein complex engineered to remain bound to DNA under suitable conditions, Panel A shows gRNA-promoted binding of variant Cas9 to a targeted DNA site. After cleavage and while Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment, panel B shows the sample was treated with an exonuclease to hydrolyze the exposed 3' or 5' ends of the DNA phosphodiester bond. After negative/enrichment selection of target DNA fragments by exonuclease destruction of all non-targeted DNA, Cas9 dissociates from DNA and releases blunt-ended double-stranded target DNA fragments of known length, as shown in panel C. Panel D depicts an optional further processing step for positive enrichment/selection of target DNA fragments by size selection. Optionally, as depicted in Figure E , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing.

5 is a schematic diagram illustrating the steps of a method for generating targeted nucleic acid fragments of known/selected length using CRISPR/Cas9 variants according to another embodiment of the present technology. Panel A shows the use of a CRISPR/Cas9 ribonucleoprotein complex engineered to remain bound to DNA under suitable conditions, where the ribonucleoprotein complex includes a capture tag. Cleavage of the double-stranded target DNA follows the variant Cas9 ribonucleoprotein complex and capture-labeled guide RNA (gRNA)-promoted binding. After cleavage and while Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment, panel B shows the sample was treated with an exonuclease to hydrolyze the exposed 3' or 5' ends of the DNA phosphodiester bond. After negative/enrichment selection of target DNA fragments by exonuclease destruction of all non-targeted DNA, and when Cas9 remains bound, Panel C shows a positive enrichment/selection process for target nucleic acid capture, which The process involves the stepwise addition of a functionalized surface capable of binding a capture label associated with the ribonucleoprotein complex as it remains bound to the target nucleic acid. Following an affinity-based enrichment step, and as depicted in Figure D , Cas9 dissociates from DNA and releases blunt-ended double-stranded target DNA fragments of known length. Panel E depicts an optional further processing step for positive enrichment/selection of target DNA fragments by size selection. Optionally, as depicted in Figure F , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing.

6 is a schematic diagram illustrating the steps of a method for generating targeted nucleic acid fragments of known/selected lengths using catalytically inactive variants of Cas9 in accordance with embodiments of the present technology. Using a catalytically inactive Cas9 ribonucleoprotein complex engineered to target and bind double-stranded DNA, Panel A shows gRNA-promoted binding of variant Cas9 to the targeted DNA site. After binding, panel B shows treatment of the sample with exonuclease to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. Catalytically inactive variants of Cas9 do not cleave target DNA, but provide exonuclease resistance such that exonuclease activity cleaves every nucleotide base until blocked by bound Cas9 complexes. After negative/enriched selection of target DNA fragments by exonuclease destruction of all non-targeted DNA, catalytically inactive Cas9 dissociates from DNA and releases double-stranded target DNA fragments of known length, as shown in Figure C Show. Panel D depicts an optional further processing step for positive enrichment/selection of target DNA fragments by size selection. Optionally, as depicted in Figure E , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing.

7 is a schematic diagram illustrating the steps of a method for generating targeted fragment sizes using catalytically inactive variants of Cas9 in accordance with another embodiment of the present technology. Panel A shows the use of a catalytically inactive variant of Cas9 in a ribonucleoprotein complex engineered to remain bound to DNA under appropriate conditions, and in which the ribonucleoprotein complex Include capture tags. Catalytically inactive variant Cas9 ribonucleoprotein complexes with capture-labeled guide RNA (gRNA)-promoted binding are followed by the addition of exonuclease to the sample to hydrolyze the exposed 3' or 5' ends of the DNA phosphodiester bond. Catalytically inactive variants of Cas9 do not cleave target DNA, but provide exonuclease resistance such that exonuclease activity cleaves every nucleotide base until blocked by bound Cas9 complexes. Panel C shows positive enrichment/enrichment of target nucleic acid capture after negative/enrichment selection of target DNA fragments by exonuclease destruction of all non-targeted DNA, and when catalytically inactive Cas9 remains bound A selection process that involves the stepwise addition of a functionalized surface capable of binding a capture label associated with the ribonucleoprotein complex as it remains bound to the target nucleic acid. Following an affinity-based enrichment step, and as depicted in Figure D , Cas9 dissociates from DNA and releases double-stranded target DNA fragments of known length. Panel E depicts an optional further processing step for positive enrichment/selection of target DNA fragments by size selection. Optionally, as depicted in Figure F , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing.

8 is a schematic diagram illustrating a target nucleic acid enrichment scheme using catalytically active and catalytically inactive Cas9 according to another embodiment of the present technology. Both catalytically active and catalytically inactive Cas9 ribonucleoprotein complexes can be targeted to desired sequences in a sample. Catalytically active Cas9 ribonucleoprotein complexes are directed to flanking regions of the target DNA region and are used to cleave the target double-stranded DNA to release blunt-ended double-stranded target DNA fragments of known length. One or more catalytically inactive ribonucleoprotein complexes with capture tags are directed to the region of the target sequence between the two site-selected cleavage sites. After cleavage of target DNA to release DNA fragments, the addition of functionalized surfaces capable of binding capture labels associated with catalytically inactive ribonucleoprotein complexes can facilitate positive enrichment/selection of target fragments.

9A and 9B are conceptual illustrations of method steps for positive enrichment/selection of target nucleic acid fragments using catalytically inactive variants of the Cas9 ribonucleoprotein complex with capture tags, according to embodiments of the present technology. Fragmented double-stranded DNA fragments (eg, mechanically sheared DNA, acoustically fragmented DNA, cell-free DNA, etc.) in a sample can be directed by targeting via the catalytically inactive Cas9 ribonucleoprotein complex in solution. Binding was positively enriched/selected (Figure 9A). Stepwise addition of a functionalized surface capable of binding a capture label associated with the ribonucleoprotein complex as it remains bound to the target nucleic acid facilitates pull-down of the desired double-stranded DNA fragment (e.g. affinity purification) while discarding non-targets oriented fragment (Figure 9B).

10 is a schematic diagram illustrating the method steps for positive enrichment/selection of target nucleic acid fragments using a catalytically inactive variant of the Cas9 ribonucleoprotein complex bearing a capture tag, according to an embodiment of the present technology. Panel A shows multiple fragmented double-stranded DNA fragments of different sizes in the sample, including molecule 2, which is too small to be reliably enriched by size selection or affinity-based methods. Panel B shows the ligation of adaptors to the 5' and 3' ends of the molecules in the sample, thereby making such DNA fragments longer in length. Panel C shows a positive enrichment/selection step for molecule 2 via target-directed binding via a catalytically inactive Cas9 ribonucleoprotein complex with a capture tag in solution, followed by affinity purification by pull-down method .

11 is a schematic diagram illustrating the steps of a method for enriching targeted nucleic acid material using a negative enrichment scheme ( panel A ) and a positive enrichment scheme ( panel B ) in accordance with an embodiment of the present technology. Panel A shows the ligation of hairpin adaptors to the 5' and 3' ends of a double-stranded target DNA molecule to generate adaptor-nucleic acid complexes without exposed ends. Adapter-nucleic acid complexes are treated with exonuclease in a negative enrichment/selection protocol to eliminate fragments of nucleic acid material and adapters with unprotected 5' and 3' ends (eg, no 4 linked phosphodi Ester-linked adaptor-nucleic acid complexes, unligated DNA, single-stranded nucleic acid material, free adaptors, etc.), as shown on the right side of panel B. Exonuclease-resistant adaptor-nucleic acid complexes can be further enriched by size selection or by target sequences (eg, CRISPR/Cas9 pulldown) (Panel B , left). The desired adaptor-target nucleic acid complexes can be further processed by amplification and/or sequencing.

Figure 12 shows an example in which a hairpin adaptor with a capture tag is ligated to a target double-stranded DNA for affinity-based enrichment and in accordance with another embodiment of the present technology.

13 is a schematic diagram illustrating method steps for positive enrichment of adaptor-target nucleic acid complexes using hairpin adaptors ( Panel A ) followed by rolling circle amplification ( Panel B and FIG. C ) and amplicon preparation steps for generating amplicons of the first and second strands of the double-stranded nucleic acid fragment at substantially the same ratio ( panel D ).

14 is a schematic diagram illustrating the steps of a method for utilizing CRISPR/Cpf1 to generate targeted nucleic acid fragments of known/selected lengths having different The 5' and 3' ligatable ends include single-stranded overhang regions of known nucleotide length and sequence. Panel A shows gRNA-promoted binding of Cpf1 at targeted DNA sites. Cpf1-directed cleavage generates staggered cuts, providing 4 (depicted) or 5 nucleotide overhangs (eg, "sticky ends"). Site-directed Cpf1 cleavage flanking the target DNA sequence generates double-stranded target DNA fragments of known length (eg, which can be enriched by size selection) with sticky ends 1 at the 5' end of the fragment and sticky ends 2 at the 3' end of the fragment ( Panel B ). Panel B further shows ligation of adaptor 1 at the 5' end of the fragment, and ligation of adaptor 2 at the 3' end of the fragment, wherein adaptor 1 and adaptor 2 comprise at least part of the cohesive ends 1 and 2 on the fragment, respectively complementary overhang sequences.

15 is a diagram illustrating steps of a method for affinity-based enrichment of target DNA fragments comprising cohesive ends (eg, such as those generated in the method of FIG. 14 ) in accordance with embodiments of the present technology Schematic. Panel A shows the stepwise addition of functionalized surfaces capable of binding sticky ends associated with target DNA fragments cleaved in solution. Once bound to the functionalized surface, affinity interactions facilitate pull-down (eg, affinity purification) of the desired double-stranded DNA fragments, while discarding non-targeted fragments, as shown in panel B.

16 is a diagram illustrating a method for affinity-based enrichment of target DNA fragments comprising sticky ends (eg, such as those generated in the method of FIG. 14 ) in accordance with another embodiment of the present technology Schematic of the steps. Panel A shows the stepwise addition of capture-labeled oligonucleotides having nucleotide sequences that are at least partially complementary to a portion of the sticky ends associated with target DNA fragments cleaved in solution. As shown in Panel B , further addition of a functionalized surface capable of binding a capture label facilitates pull-down (eg, affinity purification) of the desired double-stranded DNA fragments, while discarding non-targeted fragments.

17 is a schematic diagram showing the steps of a method for targeted fragment enrichment of nucleic acid material of known length and having different 5' and 3' ligatable ends using Cas9 nickase, according to an embodiment of the present technology, The ligable ends include long single-stranded overhangs of known nucleotide length and sequence. Panel A shows gRNA-targeted binding of paired Cas9 nickases in targeted DNA regions. Double-strand breaks can be introduced by excising the target DNA region using paired nickases, and when paired Cas9 nickases are used, long overhangs (sticky ends) are created on each cleaved end rather than blunt ends. Ends 1 and 2), as shown in Figure B. Panel C shows the stepwise addition of functionalized surfaces capable of binding long sticky ends (eg, sticky ends 1 ) associated with target DNA fragments cleaved in solution. Once bound to the functionalized surface, affinity interactions facilitate pull-down (eg, affinity purification) of the desired double-stranded DNA fragments, while discarding non-targeted fragments, as shown in panel D. Panel E shows a variation of the positive enrichment step that involves adding capture-labeled oligonucleotides with long sticky ends (eg, sticky ends) associated with target DNA fragments cleaved in solution A nucleotide sequence that is at least partially complementary to a portion of 1). Panel F shows the annealing of a second oligo strand that is at least partially complementary to a portion of the capture-labeled oligonucleotide. Enzymatic extension of the second oligo strand and ligation to the template DNA fragment generates the adaptor-target DNA complex. A further step may involve the introduction of a functionalized surface (not shown) capable of binding a capture label to facilitate pull-down (e.g., affinity purification) of the desired adaptor-dsDNA complex, while discarding non-targeted fragments .

18 is a schematic diagram illustrating a target nucleic acid enrichment scheme using catalytically inactive Cas9 according to another embodiment of the present technology. The catalytically inactive Cas9 ribonucleoprotein complex can target desired sequences in the sample. One or more catalytically inactive ribonucleoprotein complexes with one or more capture labels direct other protein complex structures to target DNA regions. Exonuclease resistance is provided when the protein complex structure covers the target DNA region. Following treatment with an exonuclease or a combination of endonuclease and exonuclease and affinity purification of the protein complex (eg, by capture tags bound to functionalized surfaces, antibody pulldown, etc.), target nucleic acid fragments Can be released from ribonucleotide complex binding.

19A and 19B are conceptual illustrations of DNA libraries and reagents prepared according to embodiments of the present technology that can be used as tools to selectively interrogate relevant DNA regions. Uniquely labeled catalytically inactive Cas9 targets multiple (eg, spaced) regions of isolated/unfragmented genomic DNA (or other large DNA fragments) (FIG. 19A). Each catalytically inactive Cas9 ribonucleoprotein includes a known oligonucleotide tag with a known sequence (eg, a code sequence) and binds to a predesigned region of the genome. When using a DNA library, the user can incrementally add one or more probes comprising the complement (eg, anti-code sequence) of the code sequence corresponding to the region of the genome of interest. A method of fragmentation can be used to fragment genomic DNA into various sizes (eg, restriction enzyme digestion, mechanical shearing, etc.). The probe includes a capture label attached or bound to it (FIG. 19B). Functionalized surfaces capable of binding capture labels can be added for affinity purification and for positive enrichment of desired genomic regions for interrogation.

20 illustrates steps of a method for affinity-based enrichment and sequencing of target DNA fragments for use with a direct digital sequencing method in accordance with an embodiment of the present technology. Panel A shows selected adaptor ligations to target DNA fragments including cohesive ends (eg, such as those generated in the methods of FIG. 14 or FIG. 17 ). Panel A further shows ligation of adaptor 1 at the 5' end of the fragment and ligation of adaptor 2 at the 3' end of the fragment, wherein adaptor 1 and adaptor 2 respectively comprise at least partially cohesive ends 1 and 2 on the fragment Complementary overhang sequence. Adaptor 1 has a Y shape and includes 5' and 3' single-stranded arms with different labels (A and B) including different properties. Adaptor 2 is a hairpin adaptor. Panel B shows the steps in a direct digital sequencing method, where label A is configured to bind to a functional surface. Label B provides physical properties (eg, charge, magnetism, etc.) such that application of an electric or magnetic field results in denaturation of the first and second strands of the double-stranded adaptor-DNA complex, followed by electrical stretching of the DNA fragments. The first and second strands remain tethered by hairpin adaptors, so that sequence information from the enriched/targeted strands provides double-stranded sequence information for error correction and other nucleic acid interrogations (eg, assessment of DNA damage, etc.).

21 illustrates steps of an affinity-based enrichment method for sequencing target DNA fragments using direct digital sequencing methods, according to another embodiment of the present technology. Panel A shows affinity-based enrichment of target DNA fragments including cohesive ends (eg, such as those generated in the methods of FIG. 14 or FIG. 17 ). As shown, the hairpin adaptor has been ligated to the 3' end of the double-stranded DNA fragment in a sequence-dependent manner. Target DNA molecules can flow over a functionalized surface (eg, with bound oligonucleotides) capable of binding sticky ends associated with cleaved target DNA fragments. Additionally, a second oligonucleotide strand comprising label B and at least partially complementary to a portion of the bound oligonucleotide is added to the solution. Annealing and ligation of the adaptor/DNA fragment components provides adaptor-target double-stranded DNA complexes that bind to a surface suitable for direct digital sequencing ( Panel B ). The application of electric or magnetic fields for the sequencing step and the electrical stretching of the adaptor-DNA complex can be performed as described, for example, in FIG. 20 .

22A illustrates nucleic acid adaptor molecules used in some embodiments of the present technology, and double-stranded adaptor-nucleic acid complexes produced by ligation of adaptor molecules to double-stranded nucleic acid fragments according to embodiments of the present technology.

22B and 22C are conceptual illustrations of various double-stranded sequencing method steps in accordance with embodiments of the present technology.

definition

For an easier understanding of the present disclosure, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

In this application, the term "a" is understood to mean "at least one" unless the context dictates otherwise. As used in this application, the term "or" can be understood to mean "and/or." In this application, the terms "comprising" and "including" may be understood to encompass the itemized components or steps, whether presented by them alone or together with one or more additional components or steps render. Where ranges are provided herein, the endpoints are included. As used in this application, the term "comprise" and variations of this term, such as "comprising" and "comprises", are not intended to exclude other additives, components, integral or steps.

About: The term "about" when used herein with reference to a value refers to a value that is, in context, similar to the reference value. In general, those skilled in the art familiar with the context will understand the relative degree of variation encompassed by "about" in this context. For example, in some embodiments, the term "about" may encompass something at 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% of the reference value %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or moieties with the reference substance. Typically, "analogs" exhibit significant structural similarity to the reference substance, such as a shared core or shared structure, but also differ in some discrete manner. In some embodiments, an analog is a substance that can be generated from a reference substance, eg, by chemical treatment of the reference substance. In some embodiments, an analog is a substance that can be generated by performing a synthetic process that is substantially similar (eg, shares multiple steps therewith) as the process for generating the reference substance. In some embodiments, the analog is or can be generated by performing a synthetic process different from that used to generate the reference substance.

Biological sample: As used herein, the term "biological sample" or "sample" generally refers to a sample obtained or derived from a relevant biological source (eg, tissue or organism or cell culture) as described herein. In some embodiments, relevant sources include organisms, such as animals or humans. In other embodiments, relevant sources include microorganisms, such as bacteria, viruses, protozoa, or fungi. In further embodiments, the relevant source may be a synthetic tissue, organism, cell culture, nucleic acid or other material. In yet further embodiments, the relevant source may be a plant-based organism. In yet another embodiment, the sample may be an environmental sample such as, for example, a water sample, a soil sample, an archaeological sample, or other sample collected from abiotic sources. In other embodiments, the sample can be a multi-organism sample (eg, a mixed-organism sample). In some embodiments, the biological sample is or includes biological tissue or fluid. In some embodiments, the biological sample can be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy; body fluids containing cells; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleura feces; lymph; gynecological fluids; skin swabs; vaginal swabs; Pap smears, buccal swabs; Fluids, aspirates; waste; bone marrow specimens; tissue biopsy specimens; fetal tissue or fluid; surgical specimens; feces, other body fluids, secretions and/or excretions; and/or cells therefrom, etc. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells from the individual from which the sample was obtained. In certain embodiments, the biological sample is a liquid biopsy sample obtained from a subject. In some embodiments, the sample is a "primary sample" obtained directly from a relevant source by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (eg, fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (eg, blood, lymph, feces, etc.) . In some embodiments, as will be clear from the context, the term "sample" refers to processing (eg, by removing one or more components of the primary sample and/or by adding to the primary sample one or more Multiple agents) formulations obtained from primary samples. For example, use semi-permeable membrane filtration. Such "treated sample" may include, for example, nucleic acid extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, separation and/or purification of certain components, or the like or protein.

Capture Tag: As used herein, the term "capture tag" (which may also be referred to as "capture tag", "capture moiety", "affinity tag", "affinity tag", "epitope tag", A "tag", "prey" moiety or chemical moiety, and other names) refers to a moiety that can be incorporated into or onto a target molecule or substrate for purification purposes. In some embodiments, the capture label is selected from the group consisting of small molecules, nucleic acids, peptides, or any uniquely bindable moiety. In some embodiments, the capture label is attached to the 5' end of the nucleic acid molecule. In some embodiments, the capture label is attached to the 3' end of the nucleic acid molecule. In some embodiments, the capture label is conjugated to a nucleotide in the internal sequence of the nucleic acid molecule but not at either end. In some embodiments, the capture label is a sequence of nucleotides within a nucleic acid molecule. In some embodiments, the capture label is selected from the group of biotin, biotin deoxythymidine dT, biotin NHS, biotin TEG, desthiobiotin NHS, digoxigenin NHS, DNP, TEG, thiols, and others. In some embodiments, capture labels include, but are not limited to, biotin, avidin, streptavidin, haptens recognized by antibodies, specific nucleic acid sequences, and magnetically attractive particles. In some embodiments, chemical modification of nucleic acid molecules (eg, Acridite ^™ -modified, adenylated, azide-modified, alkyne-modified, I-Linker ^™ -modified, etc.) can Used as a capture marker.

Cut site: Also known as "cleavage site" and "nicksite," is a bond or pair of bonds between nucleotides in a nucleic acid molecule. In the case of a double-stranded nucleic acid molecule, such as double-stranded DNA, the cleavage site may include bonds (usually phosphodiester bonds) that are immediately adjacent to each other in the double-stranded molecule, such that "blunt" ends are formed upon cleavage. The cleavage site may also include two nucleotide bonds on each single strand of the pair that are not directly opposite each other, so that when cleaved, a "sticky end" is left, thereby Regions of single-stranded nucleotides remain at the ends of the molecule. The cleavage site can be defined by a specific nucleotide sequence that can be recognized by an enzyme such as a restriction enzyme or another endonuclease with sequence recognition capabilities such as CRISPER/Cas9. The cleavage site can be within the recognition sequence of such enzymes (ie type 1 restriction enzymes) or be adjacent to them by some defined nucleotide spacer (ie type 2 restriction enzymes). The cleavage site can also be defined by the position of modified nucleotides that can be recognized by certain nucleases. For example, abasic sites can be recognized and cleaved by endonuclease VII and the enzyme FPG. Uracil bases can be recognized by the enzyme UDG and become abasic sites. When annealed to a complementary DNA sequence, ribose-containing nucleotides in the additional DNA sequence can be recognized and cleaved by RNAseH2.

Determination: Many of the methods described herein contain a step of "determination". Those of ordinary skill in the art who read this specification will understand that such "determining" can be accomplished with or through the use of any of a variety of techniques available to those of ordinary skill in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, an operation comprising a physical sample is determined. In some embodiments, the determination involves consideration and/or manipulation of data or information, eg, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining includes receiving relevant information and/or material from a source. In some embodiments, determining comprises comparing one or more characteristics of the sample or entity to a comparable reference.

Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) the production of an RNA template from a DNA sequence (eg, by transcription); (2) the processing of RNA transcripts ( For example, by splicing, editing, 5' cap formation and/or 3' end formation); (3) translation of RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

Extraction moiety: As used herein, the term "extraction moiety" (which may also be referred to as "binding partner", "affinity partner", "bait" moiety or chemical group, among other names) refers to a moiety that can A separation moiety or molecule of any type that allows affinity separation of nucleic acid bearing a capture label from nucleic acid lacking a capture label. In some embodiments, the extraction moiety is selected from the group consisting of small molecules, nucleic acids, peptides, antibodies, or any uniquely bindable moiety. The extraction moiety can be or can be linked to a solid phase or other surface for forming a functionalized surface. In some embodiments, the extraction moiety is a sequence of nucleotides attached to a surface (eg, a solid surface, beads, magnetic particles, etc.). In some embodiments, the extraction moiety is selected from the group of avidin, streptavidin, antibodies, polyhistidine tags, FLAG tags, or any chemical modification of the surface for attachment chemistry. Non-limiting examples of these latter include azide and alkyne groups or thiol azides and terminal alkynes that can form 1,2,3-triazole bonds by "click" methods and can react to fix I- Aldehyde and ketone-modified surfaces of Linker ^TM -labeled oligonucleotides, and thiol-modified surfaces can be covalently reacted with acrylate-modified oligonucleotides.

Functionalized surface: As used herein, the term "functionalized surface" refers to a solid surface, bead, or another immobilized structure capable of binding or immobilizing a capture label. In some embodiments, the functionalized surface includes extraction moieties capable of binding capture labels. In some embodiments, the extraction portion is directly attached to the surface. In some embodiments, chemical modification of the surface acts as an extraction moiety. In some embodiments, functionalized surfaces can include controlled pore glass (CPG), magnetic porous glass (MPG), and other glass or non-glass surfaces. Chemical functionalization can include ketone modifications, aldehyde modifications, thiol modifications, azide modifications, and alkyne modifications, among others. In some embodiments, the functionalized surface and the oligonucleotide for adaptor synthesis are linked using one or more of a set of immobilization chemistries that form amide bonds, alkylamine bonds, Thiourea bonds, diazo bonds, hydrazine bonds, and other surface chemistries. In some embodiments, the functionalized surface is attached to the oligonucleotide for adaptor synthesis using one or more of a set of reagents comprising EDAC, NHS, sodium periodate, glutaraldehyde , pyridyl disulfide, nitrous acid, biotin, and other linking reagents.

gRNA: As used herein, "gRNA" or "guide RNA" refers to a short RNA molecule comprising an endonuclease suitable for targeting (eg a Cas enzyme such as Cas9 or Cpf1 or another with similar properties ribonucleoproteins, etc.), the targeted endonucleases bind to substantially target-specific sequences that facilitate cleavage of specific regions of DNA or RNA.

Nucleic acid: As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and/or substances that are or can be incorporated into oligonucleotide chains through phosphodiester linkages. As will be clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (eg, nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to comprising Oligonucleotide chains of single nucleic acid residues. In some embodiments, "nucleic acid" is or includes RNA; in some embodiments, "nucleic acid" is or includes DNA. In some embodiments, the nucleic acid is, comprises or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises or consists of one or more "peptide nucleic acids" known in the art and having peptide bonds in the backbone rather than phosphates Diester linkages are considered to be within the scope of the present technology. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages instead of phosphodiester linkages. In some embodiments, the nucleic acid is, includes, or consists of one or more natural nucleosides (eg, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, includes, or consists of one or more nucleoside analogs (eg, 2-aminoadenosine, 2-thiopyrimidine, inosine, pyrrolopyrimidine, 3-methyladenosine , 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine glycoside, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxo adenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, hexose or locked nucleic acid). In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid can be a non-protein-coding RNA product, such as a microRNA, ribosomal RNA, or CRISPER/Cas9 guide RNA. In some embodiments, the nucleic acid functions as a regulator in the genome. In some embodiments, the nucleic acid is not from the genome. In some embodiments, the nucleic acid comprises an intergenic sequence. In some embodiments, the nucleic acid is derived from an extrachromosomal element or a non-nuclear genome (mitochondria, chloroplast, etc.). In some embodiments, nucleic acids are prepared by one or more of isolation from natural sources, enzymatic synthesis (in vivo or in vitro) by polymerization of complementary templates, replication in recombinant cells or systems, and chemical synthesis. In some embodiments, the nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 , 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is partially or fully single-stranded; in some embodiments, the nucleic acid is partially or fully double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element encoding a polypeptide, or is the complement of a sequence encoding a polypeptide. In some embodiments, the nucleic acid has enzymatic activity. In some embodiments, the nucleic acid functions mechanically, eg, in a ribonucleoprotein complex or transfer RNA. In some embodiments, the nucleic acid functions as an adaptor. In some embodiments, nucleic acids can be used for data storage. In some embodiments, nucleic acids can be chemically synthesized in vitro.

Reference: As used herein, describes a standard or control against which to compare. For example, in some embodiments, a related agent, animal, individual, population, sample, sequence or value is compared to a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, the reference or control is tested and/or determined substantially concurrently with the relevant test or determination. In some embodiments, the reference or control is a historical reference or control, optionally contained in a tangible medium. Typically, as will be understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances that are comparable to the conditions or circumstances being assessed. Those skilled in the art will understand when sufficient similarity exists to justify reliance and/or comparison with a particular potential reference or control.

Single Molecule Identifier (SMI): As used herein, the term "Single Molecule Identifier" or "SMI" (which may be referred to as "tag", "barcode", "molecular barcode", "unique molecular identifier" ” or “UMI,” and other names) refers to any material (eg, nucleotide sequence, nucleic acid molecular signature) capable of distinguishing individual molecules within a large heterogeneous population of molecules. In some embodiments, the SMI may be or include an exogenously applied SMI. In some embodiments, the exogenously applied SMI may be or include a degenerate or semi-degenerate sequence. In some embodiments, a substantially degenerate SMI may be referred to as a random unique molecular identifier (R-UMI). In some embodiments, the SMI may include codes (eg, nucleic acid sequences) from within a pool of known codes. In some embodiments, the predefined SMI codes are referred to as Defined Unique Molecular Identifiers (D-UMIs). In some embodiments, the SMI can be or include an endogenous SMI. In some embodiments, an endogenous SMI can be or include information related to a specific cleavage site of the target sequence or a feature associated with the end of a single molecule that includes the target sequence. In some embodiments, SMI may involve sequence variation in a nucleic acid molecule caused by random or semi-random damage, chemical modification, enzymatic modification, or other modification to the nucleic acid molecule. In some embodiments, the modification can be the deamination of methylcytosine. In some embodiments, modifications may require nucleic acid nicking sites. In some embodiments, the SMI can include exogenous and endogenous elements. In some embodiments, the SMI may include physically adjacent SMI elements. In some embodiments, the SMI elements can be spatially distinct within the molecule. In some embodiments, the SMI can be non-nucleic acid. In some embodiments, an SMI may include two or more different types of SMI information. Various embodiments of SMI are further disclosed in International Patent Publication No. WO2017/100441, the entire contents of which are incorporated herein by reference.

Strand-Defining Element (SDE): As used herein, the term "strand-defining element" or "SDE" refers to any material that allows a particular strand of double-stranded nucleic acid material to be identified and thus distinguished from another/complementary strand (eg, Any material that makes the amplification products of each of two single-stranded nucleic acids generated from a target double-stranded nucleic acid substantially distinguishable from each other after sequencing or other nucleic acid interrogation). In some embodiments, the SDE can be or include one or more fragments of substantially non-complementary sequences in the adaptor sequence. In particular embodiments, fragments of substantially non-complementary sequences in the adaptor sequence can be provided by adaptor molecules comprising Y-shapes or "loops". In other embodiments, fragments of substantially non-complementary sequences in the adaptor sequence may form unpaired "bubbles" in the middle of adjacent complementary sequences in the adaptor sequence. In other embodiments, the SDE may comprise nucleic acid modifications. In some embodiments, the SDE may include physical separation of pairs of chains into physically separate reaction chambers. In some embodiments, the SDE can include chemical modifications. In some embodiments, SDEs can include modified nucleic acids. In some embodiments, SDE may involve sequence variation in a nucleic acid molecule caused by random or semi-random damage, chemical modification, enzymatic modification, or other modifications to the nucleic acid molecule. In some embodiments, the modification can be the deamination of methylcytosine. In some embodiments, modifications may require nucleic acid nicking sites. Various embodiments of SDE are further disclosed in International Patent Publication No. WO2017/100441, the entire contents of which are incorporated herein by reference.

Subject: As used herein, the term "subject" refers to an organism, typically a mammal (eg, a human, including in some embodiments a prenatal human form). In some embodiments, the subject suffers from a related disease, disorder or condition. In some embodiments, the subject is susceptible to a disease, disorder or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder or condition. In some embodiments, the subject is a human having one or more characteristics of susceptibility or risk characteristics for a disease, disorder or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and/or therapy is administered and/or has been administered.

Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or nearly all of the extent or degree of the characteristic or property concerned. One of ordinary skill in the biological arts will understand that biological and chemical phenomena are rarely, if ever, accomplished and/or progressed to completion or absolute results are achieved or avoided. Thus, the term "substantially" is used herein to capture the underlying lack of integrity inherent in many biological and chemical phenomena.

Detailed ways

The present technology generally relates to methods for enriching nucleic acid material for sequencing applications and other interrogation of nucleic acid material and related reagents for such methods. Some embodiments of this technology relate to enriching one or more relevant regions in nucleic acid material for sequencing applications, such as double-stranded sequencing applications and other sequencing applications for achieving high precision sequencing reads. For example, various embodiments of the present technology comprise selectively enriching nucleic acid material (eg, genomic DNA material) in regions of interest, and performing a double-stranded sequencing method to provide error-corrected sequence reads of the enriched nucleic acid material. Further examples of the present technology relate to methods for double-stranded sequencing methods or other sequencing methods (eg, single consensus sequencing methods, Hyb&Seq ^™ sequencing methods, nanopore sequencing methods, etc.) for nucleic acid material enriched for regions of interest. In various embodiments, the enrichment of nucleic acid material is provided at a faster rate (eg, with fewer steps) and at a lower cost (eg, using fewer reagents), comprising enriching the nucleic acid material to a relevant area and result in an increase in the required data. Various aspects of the present technology have numerous applications in preclinical and clinical testing and diagnostics, as well as other applications.

Double-stranded sequencing (DS) is a method for generating error-corrected nucleic acid sequence reads from double-stranded nucleic acid molecules. In certain aspects of the technology, DS can be used to independently sequence two strands of a single nucleic acid molecule in such a way that during massively parallel sequencing, derived sequence reads can be identified as originating from the same double strand Nucleic acid parent molecules, but can also be distinguished from each other as distinguishable entities after sequencing. The resulting sequence reads from each strand are then compared for the purpose of obtaining an error-corrected sequence (referred to as a double-stranded consensus sequence) of the original double-stranded nucleic acid molecule. The process of DS makes it possible to confirm whether one or both strands of the original double-stranded nucleic acid molecule are represented in the generated sequencing data used to form the double-stranded consensus sequence.

Standard next-generation sequencing has an error rate of about 1/100-1/1000, and when fewer than 1/100-1/1000 molecules carry sequence variants, their presence is masked by the background error rate of the sequencing process. On the other hand, DS can accurately detect extremely low frequency variants due to the high degree of error correction obtained. The high degree of error correction provided by DS's strand comparison technology reduces sequencing errors of double-stranded nucleic acid molecules by orders of magnitude compared to standard next-generation sequencing methods. This reduction in error increases the accuracy of sequencing for nearly all types of sequences, but may be particularly well suited for biochemically challenging sequences that are well known in the art to be particularly error-prone, or where the population of molecules being sequenced is heterogeneous. Qualitative (ie, small subsets of molecules carry sequence variants that other molecules do not carry). A non-limiting example of such a type of sequence is a homopolymer or other microsatellites/short tandem repeats. Another non-limiting example of an error-prone sequence that benefits from DS error correction is a molecule that has been destroyed, for example, by heat, radiation, mechanical stress, or various chemical exposures that result from being destroyed by a or various chemical adducts that are error-prone during replication of nucleotide polymerases, and those that create single-stranded DNA or as gaps and gaps at the ends of the molecule. In highly damaged DNA (oxidation, deamination, etc.) that occurs through fixation processes (i.e. FFPE in clinical pathology) or ancient DNA or in forensic applications where the material has been exposed to harsh chemicals or environments, Double-stranded sequencing is particularly useful for reducing the high level of error caused by damage.

In a further embodiment, DS can also be used to accurately detect minority sequence variants in a population of double-stranded nucleic acid molecules. A non-limiting example of this application is the detection of a small number of cancer-derived DNA molecules in a larger number of unmutated molecules from non-cancerous tissue in a subject. DS is also well suited for precise genotyping of difficult-to-sequence regions of the genome (homopolymers, microsatellites, G-quadruplexes, etc.), where the error rate of standard sequencing is particularly high. Another non-limiting application of rare variant detection by DS is the early detection of DNA damage caused by genotoxin exposure. Another non-limiting application of DS is the detection of mutations generated by genotoxic or non-genotoxic carcinogens by observing gene clones that exhibit driver mutations. Yet a further non-limiting application for the precise detection of few sequence variants is the generation of mutagenic markers associated with genotoxins. Additional non-limiting examples of the utility of DS can be found in Salk et al, Nature Reviews Genetics 2018, PMID 29576615 (which is incorporated herein by reference in its entirety).

分析系统(NanoString^TMTechnologies；Seattle,WA)。包含分子“条形码”的方法和试剂以及适合于NanoString^TM技术的设备在例如美国专利公开第2010/0112710号、第2010/0047924号、第2010/0015607号(每一个的全部内容通过引用并入本文)中进一步描述。Various embodiments related to enrichment of nucleic acid material for sequencing applications as well as other nucleic acid material interrogation have utility in single molecule sequencing applications and direct digital sequencing methods. In some embodiments, techniques using single-molecule hybridization with barcoded probes can be used to characterize and/or quantify genomic regions. In general, such techniques use molecular "barcoding" and single-molecule imaging to detect and count specific nucleic acid targets in a single reaction without amplification. Typically, each color-coded barcode is attached to a single target-specific probe corresponding to the relevant genomic region. They are mixed with controls to form a multiplexed code set. In some embodiments, two probes are used to hybridize each individual target nucleic acid. In certain arrangements, the reporter probe carries the signal and the capture probe allows the complex to be immobilized for data collection. After hybridization, excess probes are removed and the immobilized probe/target complexes can be analyzed by a digital analyzer for data collection. Color codes for each target molecule (eg, associated genomic region) are counted and listed. A suitable digital analyzer contains

Analysis system (NanoString ^™ Technologies; Seattle, WA). Methods and reagents comprising molecular "barcodes" and devices suitable for NanoString ^™ technology are described in, eg, US Patent Publication Nos. 2010/0112710, 2010/0047924, 2010/0015607 (the entire contents of each are incorporated herein by reference) ) are further described in.

Direct digital sequencing (DDS) technology encompasses methods for providing highly accurate single-molecule sequencing that simultaneously captures and directly sequences DNA and RNA for a variety of research, diagnostic, and other applications. DDS provides both short and long sequencing reads without the need for library creation or amplification steps, and is described, for example, in International Patent Publication No. WO 2016/081740, which is incorporated herein by reference. Typically, direct sequencing of nucleic acid targets is accomplished by hybridizing fluorescent molecular barcodes to native nucleic acid targets. As further described in US Pat. No. 7,919,237, and as available from NanoString ^™ Technologies, Inc. (Seattle, WA), oligomers that are extensions of targeted nucleotide sequences are stretched by electrostretching techniques, The monomers are spatially separated, where each monomer is attached to a unique label. Thus, the pattern of labeled monomers can be used to identify barcodes on oligomeric tags.

Furthermore, various embodiments related to the enrichment of nucleic acid material have utility in other forms of characterization and/or quantification of nucleic acid material, which are known in the art. For example, determination of the presence or absence of genomic mutations, quantification of DNA variants, DNA or RNA copy number, and characterization of nucleic acid material for other applications can benefit from selective enrichment of target nucleic acid material as provided herein. Examples of some methods include, but are not limited to, single-molecule sequencing (eg, single-molecule real-time sequencing, nanopore sequencing, high-throughput sequencing, or next-generation sequencing (NGS), etc.), digital PCR, bridging PCR, emulsion PCR, semiconductor sequencing, and the like. One of ordinary skill in the art will recognize other nucleic acid interrogation methods and techniques that may be suitable for interrogation and/or benefit from enriched nucleic acid material.

Methods of incorporating DS, as well as other sequencing modalities, can involve ligating one or more sequencing adaptors to a target double-stranded nucleic acid molecule to generate a double-stranded target nucleic acid complex. Such adaptor molecules may comprise one or more of a variety of features suitable for MPS platforms, such as, for example, sequencing primer recognition sites, amplification primer recognition sites, barcodes (eg, single molecule identifier (SMI) sequences, index sequences, single-stranded portions, double-stranded portions, strand distinguishing elements or features, etc.). The use of highly pure sequencing adapters for DS or any next-generation sequencing technology is important for obtaining high-quality reproducible data and maximizing the sequence yield of the sample (i.e. the relative percentage of input molecules converted to independent sequence reads) . This is particularly important for DS as both strands of the original double-stranded molecule need to be successfully recovered.

Regarding the efficiency of the DS process or other high-precision sequencing modalities, this paper further describes two types of efficiencies: conversion efficiency and workflow efficiency. For purposes of discussing the efficiency of DS, transformation efficiency can be defined as the fraction of unique nucleic acid molecules input into a sequencing library preparation reaction, resulting in at least one double-stranded consensus sequence read. Workflow efficiency may be related to the amount of time required to perform these steps to generate double-stranded sequencing libraries and/or targeted enrichment of related sequences, the relative number of steps and/or the relative inefficiency of the financial cost of reagents/materials related.

In some cases, one or both of conversion efficiency and workflow efficiency constraints may limit the usefulness of high precision DS in some applications where it would otherwise be well suited. For example, a low transformation efficiency will lead to situations where the copy number of the target double-stranded nucleic acid is limited, which may result in a lower than desired amount of sequence information being produced. Non-limiting examples of this concept include DNA from circulating tumor cells or cell-free DNA from tumors, or DNA from prenatal infants shed into body fluids such as plasma and mixed with excess DNA from other tissues. Although DS typically has the accuracy of being able to resolve one mutated molecule out of over a hundred thousand unmutated molecules, for example, if only 10,000 molecules are available in the sample, and even if these are converted to double-stranded consensus reads, the ideal efficiency is 100%, then the lowest mutation frequency that can be measured would be 1/(10,000*100%)=1/10,000. As a clinical diagnosis, it may be important to have maximum sensitivity to detect low-level signals of cancer or therapy-related mutations, and thus relatively low transformation efficiencies would be undesirable in this case. Similarly, in forensic applications, often very little DNA is available for testing. The greatest transformation efficiency is achieved when only recovering from a crime scene or natural disaster scene to nanogram or picogram quantities, and where DNA from multiple individuals is mixed together, is possible for being able to detect the presence of DNA from all individuals in the mix is important.

In some cases, workflow inefficiencies can be similarly challenging for certain nucleic acid interrogation applications. A non-limiting example of this is clinical microbiology testing. Sometimes there is a need to rapidly test the properties of one or more infectious organisms, for example, microbial or polymicrobial bloodstream infections, some of which are resistant to specific antibiotics based on the unique genetic variants they carry, but cultured And the time required to empirically determine the antibiotic susceptibility of an infectious organism is much longer than the time required to make a therapeutic decision about the antibiotic to be used for treatment. DNA sequencing of DNA from blood (or other infected tissues or body fluids) has the potential to be more rapid and, for example, among other high-precision sequencing methods, DS can very accurately detect therapeutically important in infected populations based on DNA markers few variants. Since workflow turnaround time for data generation is critical in determining treatment options (eg, as in the examples used herein), applications that increase the speed to data output would also be desirable.

Further disclosed herein are methods and compositions for targeted nucleic acid sequence enrichment for a variety of nucleic acid material interrogation applications. In particular, some aspects of the present technology relate to methods and compositions for the enrichment of targeted nucleic acid material, and the use of such enrichment in error-corrected nucleic acid sequencing applications, the cost, conversion of sequenced molecules and provides improvements in the time efficiency of generating labeled molecules for targeted ultra-high precision sequencing.

I. Selected Examples of Methods and Reagents for Enrichment of Nucleic Acid Materials

In some embodiments, provided methods provide targeted enrichment strategies compatible with the use of molecular barcodes for error correction. Other embodiments provide methods for non-amplification-based targeted enrichment strategies compatible with DDS and other sequencing strategies (eg, single-molecule sequencing modes and interrogation) that do not use molecular barcodes.

In some embodiments, it is advantageous to process the nucleic acid material in order to increase the efficiency, accuracy and/or speed of the sequencing process. According to further aspects of the present technology, the efficiency of DS can be increased, for example, by fragmentation of targeted nucleic acids. Traditionally, fragmentation of nucleic acids (eg, genomes, mitochondria, plasmids, etc.) is accomplished by physical shearing (eg, sonication) or relatively non-sequence-specific enzymatic methods that utilize mixtures of enzymes to cleave DNA phosphodiester bonds. The result of any of the above methods is a sample in which intact nucleic acid material (eg, genomic DNA (gDNA)) is reduced to a mixture of random or semi-random sized nucleic acid fragments. Although effective, these methods generate nucleic acid fragments of variable size, which can lead to amplification bias (e.g., short fragments tend to be PCR amplified more efficiently than long fragments, and may be more prone to cluster amplification during polymerase clone formation) increase) and uneven sequencing depth. For example, Figure 1 is a graph plotting the relationship between nucleic acid insert size and the resulting family size after amplification of a population of DNA molecules labeled with different molecular barcodes during library preparation. As shown in Figure 1, because shorter fragments tend to amplify preferentially, on average a greater number of copies of each of these shorter fragments are generated and sequenced, providing a disproportionate amount of sequencing depth for these regions s level.

Furthermore, for longer fragments, parts of DNA between the limits of sequencing reads (or between the ends of paired-end sequencing reads) cannot be interrogated if they extend beyond the maximum read length of the sequencing platform and are "dark" , although it was successfully ligated, amplified and captured (Fig. 2A). Also, for short fragments, and when paired-end sequencing is used, overlapping reads from two reads covering the same sequence in the middle of the molecule provide redundant information and are cost-inefficient (Figure 2B). Random or semi-random nucleic acid fragmentation can also lead to unpredictable breakpoints in the target molecule that generate fragments that may not be complementary or have reduced complementarity to the bait strand used for hybrid capture, thereby reducing target capture efficiency . Random or semi-random fragmentation may also disrupt related sequences and/or result in very small or very large fragments that are lost during other stages of library preparation and may reduce data yield and efficiency.

Another problem with many methods of random fragmentation, especially mechanical or acoustic methods, is that they introduce damage beyond double-strand breaks, which can render partially double-stranded DNA no longer double-stranded. For example, mechanical shearing can create 3' or 5' overhangs at the ends of the molecule and single-stranded gaps or gaps in the middle of the molecule. These single-stranded moieties suitable for adaptor ligation (such as a cocktail of "end repair" enzymes) are used to artificially double-stranded again, and this can be a source of artificial error (such as, for example, "end repair" as described herein) Pseudodouble-stranded molecules"). In many embodiments, it is optimal to maximize the amount of associated double-stranded nucleic acid that remains in its native double-stranded form during processing. In addition, the high energies involved in many methods of random or semi-random mechanical fragmentation increase the abundance of DNA damage, such as oxidation, deamination, or other adduct formation during amplification or sequencing May be mutagenic or repressive, and may introduce artefact base calls or reduced signal. Some random or semi-random enzymatic fragmentation methods can similarly leave mutagenic or closed "scars" at the site of partial cleavage.

Furthermore, for DS processing, the two strands of the original target nucleic acid molecule must be successfully ligated. For example, in embodiments where adaptors are ligated to the 5' and 3' ends of the molecule, four phosphodiester linkages must be successfully created. If one of these bonds cannot be formed, it is impossible to amplify and sequence both strands of the molecule. As noted above, failure to form the necessary bonds can be due to a variety of reasons (including, for example, damage to the ends of the target double-stranded nucleic acid molecule, incomplete end repair or tailing of library fragments, incomplete synthesis or damaged adaptors) Molecular, contaminating ligation, or previous reactions, for example, have undesired enzymatic activity (e.g., exonuclease activity that can destroy the ligable ends of adapters or library fragments, or degradation of the ligase, rendering it multi-level catalytically active) invalid)) and other reasons. Damage to the ends of library fragments can be particularly common during high-energy sonication or other mechanical DNA fragmentation.

In addition to successful adaptor ligation, both the first and second strands of the adaptor-target nucleic acid complex must be amplifiable to achieve double-stranded sequence accuracy. For example, if a particular strand of a target nucleic acid molecule is nicked or destroyed in a manner that the polymerase cannot traverse, amplification of that particular strand will not occur and double-stranded consensus reads cannot be generated. As non-limiting examples, non-traversable lesions can be introduced by sonication DNA fragmentation, enzymatic steps of high temperature or elongation, or single-strand nicking activity in library preparation.

Thus, in other applications, DS may benefit from increased efficiency by utilizing one or more methods for enriching target nucleic acid in a sample comprising enriching the target nucleic acid material prior to the amplification step. Regardless of the underlying method, detection of rare nucleic acid variants requires screening of large numbers of molecules; however, the more molecules (ie, genomic equivalents) that are prepared simultaneously into a library, the lower the relative efficiency of the process.

Various aspects of the present technology provide methods, reagents and nucleic acid libraries and kits for enriching nucleic acid material for sequencing applications and other nucleic acid interrogations. Additional aspects of the present technology provide solutions to improve the conversion and workflow efficiency of DS and other sequencing modalities to overcome most of the limitations listed above.

Some aspects of the present technology relate to methods for enriching regions of interest using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) programmable endonuclease system. In other aspects, CRISPER-like or other programmable endonucleases such as zinc finger nucleases, TALEN nucleases or other sequence specific endonucleases such as homing endonucleases or simple restriction nucleases or derivatives thereof Can be used alone or in combination as part of the disclosed technology.

In particular, CRISPR/Cas9 (or other programmable or non-programmable endonucleases or a combination thereof) can be used to selectively cleave the nucleic acid backbone in one or more defined or semi-defined regions in order to recover from relatively Functionally excising one or more relevant sequence regions in a long nucleic acid molecule, wherein the excised target region is designed to have one or more predetermined or substantially predetermined lengths to enable use in sequencing applications such as DS) is enriched for one or more relevant nucleic acid target regions by size selection prior to library preparation. In other embodiments, CRISPR/Cas9 (or other programmable endonucleases or non-programmable endonucleases or combinations thereof) can be used to selectively excise one or more relevant sequence regions, wherein the excised The target region is designed to have a sequence of substantially predetermined lengths and overhangs. These programmable endonucleases can be used alone or in combination with other forms of targeted nucleases such as restriction endonucleases or other enzymatic or non-enzymatic methods for cleaving nucleic acids.

In some embodiments, provided methods can comprise the steps of: providing nucleic acid material, cleaving the nucleic acid material with a targeted endonuclease (eg, a ribonucleoprotein complex) such that one or more substantially predetermined lengths The target region is isolated or enriched from the rest of the nucleic acid material, and the cleaved target region is analyzed. In other embodiments, one or more cleaved regions can be negatively enriched (ie, depleted) from the remainder of the nucleic acid material and not analyzed. In some embodiments, the provided methods can further comprise ligating at least one SMI and/or adaptor sequence to at least one of the 5' or 3' end of the cleaved target region of predetermined length. In some embodiments, analysis may be or include quantification and/or sequencing.

In some embodiments, quantification may be or include spectrophotometric analysis, real-time PCR, and/or fluorescence-based quantification (eg, using fluorescent dye labeling). In some embodiments, sequencing can be or include Sanger sequencing, shotgun sequencing, bridge PCR, nanopore sequencing, single molecule real-time sequencing, ion torrent sequencing, pyrosequencing, digital sequencing (eg, digital barcode-based sequencing), Sequencing by ligation, polymerase cloning-based sequencing, current-based sequencing (eg, tunneling current), sequencing by mass spectrometry, microfluidics-based sequencing, Illumina sequencing, next-generation sequencing, massively parallel sequencing, and any combination thereof .

In some embodiments, the targeted endonuclease is or includes a CRISPR-associated (Cas) enzyme (eg, Cas9 or Cpf1) or other ribonucleoprotein complexes, homing endonucleases, zinc finger nucleases, transcription At least one of activator-like effector nucleases (TALENs), arginine nucleases, megaTAL nucleases, meganucleases, and/or restriction endonucleases. In some embodiments, more than one targeted endonuclease may be used (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, targeted nucleases can be used to cleave more than one predetermined length (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of potential targets area. In some embodiments in which there is more than one target region of predetermined length, each target region may have the same (or substantially the same) length. In some embodiments in which there is more than one target region of predetermined length, at least two target regions of predetermined length differ in length (eg, a first target region has a length of 100 bp and a second target region has a length of 1,000 bp length).

The present disclosure also provides methods and reagents for affinity-based enrichment of target nucleic acid materials. In some embodiments comprising such methods, one or more capture labels or moieties can be used to enrich/select desired target nucleic acid material from samples including genomic material, non-target nucleic acid material, contamination nucleic acid material, nucleic acid material from mixed samples, cfDNA material, etc. For example, some embodiments include the use of one or more capture labels/moieties for positive enrichment/selection of desired target nucleic acid material (eg, including target sequences or associated genomic regions, associated in unfragmented genomic DNA) Fragments of targeted genomic regions). In other embodiments, capture markers can be used for negative enrichment/selection to exclude or reduce the abundance of undesired genomic material.

For example, in some embodiments comprising positive enrichment, the adaptor oligonucleotide can have a capture label that is or includes an attached chemical moiety (eg, biotin) that can be used to pass the Capture in multiple subsequent purification steps, e.g. by extracting moieties (eg streptavidin) bound to functionalized surfaces (eg paramagnetic beads or other forms of beads) to isolate or isolate the desired adaptor- Nucleic acid complexes. In some embodiments involving negative enrichment, a capture label that is or includes an attached chemical moiety (eg, biotin) can be used for capture by in one or more subsequent purification steps, such as by binding to a functionalized surface Extract fractions (eg, streptavidin) (eg, paramagnetic beads or other forms of beads) to purify or isolate undesired genomic material ligated or attached to adapters (or other probes including capture labels) (eg, off-target nucleic acid fragments, etc.).

Size-based enrichment of nucleic acid material

In some embodiments, provided methods and compositions utilize targeted endonucleases (eg, ribonucleoprotein complexes (CRISPR-associated endonucleases such as Cas9, Cpf1), homing endonucleases Enzymes, zinc finger nucleases, TALENs, arginine nucleases, meganucleases, restriction endonucleases, and/or meganucleases (eg, megaTAL nucleases, etc., or combinations thereof) or capable of cleaving nucleic acid material (eg, , one or more restriction enzymes) other techniques to excise the relevant target sequence at the optimal fragment size for sequencing. In some embodiments, the targeted endonuclease has the ability to specifically and selectively excise a precise sequence region of interest. Bias can be significantly reduced by preselecting cleavage sites, eg, using programmable endonucleases (eg, CRISPR-associated (Cas) enzyme/guide RNA complexes) that generate fragments of predetermined and substantially uniform size and the presence of non-informative readings. Furthermore, due to the size difference between the excised fragments and the remaining uncleaved DNA, a size selection step (as described further below) can be performed to remove large off-target regions, thereby pre-enriching the sample prior to any further processing steps . The need for end repair steps may also be reduced or eliminated, saving time and risk of false duplex challenges, and in some cases, the need for computational trimming of data near the ends of the molecule, thereby increasing efficiency. Therefore, an additional advantage of targeted cleavage is the potential to reduce gaps or nucleic acid adducts or other forms of damage caused by mechanical fragmentation methods.

The method known as CRISPR-DS allows for very high on-target enrichment (which can reduce the need for subsequent hybridization capture steps), which can significantly reduce time and cost as well as improve transformation efficiency. 3 is a schematic diagram illustrating the steps of a method for generating targeted fragment sizes using CRISPR/Cas9 according to an embodiment of the present technology. For example, CRISPR/Cas9 can be used for gRNA-facilitated binding via Cas9, at one or more specific sites (eg, protospacer-adjacent motif or "PAM" sites) within the target sequence (Figure 3, Panel A ) ) is cut. Cas9-directed cleavage releases blunt-ended double-stranded target DNA fragments of known length, as shown in panel B. Figure 3, panel C depicts further processing steps for positive enrichment/selection of target DNA fragments by size selection. One method of isolating excised target moieties involves the use of SPRI/Ampure beads and magnetic purification to remove high molecular weight DNA while leaving predetermined shorter fragments. In other embodiments, various size selection methods (including, but not limited to, gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, and/or filtration purification methods), as well as other methods, can be used to isolate unwanted DNA from Fragments and other high molecular weight genomic DNA (if applicable) are isolated from excised portions of predetermined lengths. Following size selection, the CRISPR-DS method may contain steps consistent with the DS method steps, including A-tailing (CRISPR/Cas9 excision leaves blunt ends), ligation of adaptors (eg DS adaptors), double-stranded amplification, An optional capture step and amplification (eg, PCR), then each strand is sequenced and a double-stranded consensus sequence is generated. In addition to improving workflow efficiency, CRISPR-based size selection/target enrichment provides optimal fragment lengths for efficient amplification and sequencing steps. Various aspects of CRISPR-DS are disclosed in International Patent Publication No. WO/2018/175997, the entire contents of which are incorporated herein by reference.

In certain embodiments, CRISPR-DS addresses a number of common problems associated with NGS, including, for example, inefficient target enrichment, which can be optimized by CRISPR-based size selection; sequencing errors, which can be eliminated using DS technology , to generate error-corrected double-stranded consensus sequences; and uneven fragment size, which was reduced by predesigned CRISPR/Cas9 fragmentation. As understood by those of skill in the art, as described herein, CRISPR-DS may have applications for sensitive identification of mutations in situations where the sample is DNA-constrained, such as forensic and early cancer detection applications, among others.

In vitro digestion of DNA material with Cas9 nuclease utilizes the formation of ribonucleoprotein complexes that recognize and cleave predetermined sites (eg PAM sites, Figure 3, Panel A ). This complex is formed by guide RNA ("gRNA" eg crRNA+tracrRNA) and Cas9. For multiple cleavage, gRNAs can be complexed by pooling all crRNAs and then complexing with tracrRNA, or by complexing each crRNA and tracrRNA separately and then pooling. In some embodiments, the second option may be preferred because it eliminates competition between crRNAs. Other CRISPER systems using different Cas proteins may rely on different PAM motif sequences, or do not require PAM motif sequences, or rely on other forms of nucleic acid sequences to direct the delivery of nucleases to targeted nucleic acid regions.

In some embodiments, the nucleic acid material comprises nucleic acid molecules of substantially uniform length. In some embodiments, the substantially uniform length is about 1 to 1,000,000 bases. For example, in some embodiments, the substantially uniform length may be at least 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 15; 20; 25; 30; 35; 40; 50; 60; 70; 80; 90; 100; 120; 150; 200; 300; 400; 500; 600; 700; 800; 900; 9000; 10,000; 15,000; 20,000; 30,000; 40,000; or 50,000 bases in length. In some embodiments, the substantially uniform length may be at most 60,000; 70,000; 80,000; 90,000; 100,000; 120,000; 150,000; As a specific non-limiting example, in some embodiments, the substantially uniform length is about 100 to about 500 bases. In some embodiments, a size selection step, such as those described herein, can be performed before any particular amplification step. In some embodiments, any particular amplification step can be followed by a size selection step, such as those described herein. In some embodiments, a size selection step, such as described herein, may be followed by additional steps, such as a digestion step and/or another size selection step. In some embodiments, size selection can be performed before or after the step of ligating adaptors. In some embodiments, the size selection may be performed concurrently with the cutting step. In some embodiments, size selection may be performed after the cutting step.

In addition to the use of targeted endonucleases, any other suitable application method that results in nucleic acid molecules of substantially uniform length can be used. By way of non-limiting example, such methods may be or include the use of one or more of the following: agarose or other gels, gel electrophoresis, affinity columns, HPLC, PAGE, filtration, gel filtration, exchange Chromatography, SPRI/Ampure-type beads, or any other suitable method that will be recognized by those skilled in the art.

In some embodiments, processing the nucleic acid material to produce nucleic acid molecules of substantially uniform length (or mass) can be used to recover one or more desired target regions from a sample (eg, a relevant target sequence). In some embodiments, processing nucleic acid material to produce nucleic acid molecules of substantially uniform length (or mass) can be used to exclude samples (eg, nucleic acid material from an undesired species of the same species or an undesired subject) specific part of the . In some embodiments, the nucleic acid material may be present in a variety of sizes (eg, not in substantially uniform length or mass).

In some embodiments, more than one targeted endonuclease or other method can be used to provide nucleic acid molecules of substantially uniform length (eg, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more). In some embodiments, a targeted nuclease can be used to cleave more than one potential target region (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of a nucleic acid material ). In some embodiments in which more than one target region of nucleic acid material is present, each target region may have the same (or substantially the same) length. In some embodiments in which more than one target region of nucleic acid material is present, at least two target regions of known length differ in length (eg, the first target region has a length of 100 bp and the second target region has a length of 1,000 bp length).

In some embodiments, multiple targeted endonucleases (eg, programmable endonucleases) can be used in combination to fragment multiple regions of a related target nucleic acid. In some embodiments, one or more programmable targeted endonucleases can be used in combination with other targeted nucleases. In some embodiments, one or more targeted endonucleases can be used in combination with random or semi-random nucleases. In some embodiments, one or more targeted endonucleases can be used in combination with other random or semi-random methods of nucleic acid fragmentation, such as mechanical or acoustic shearing. In some embodiments, it may be advantageous to perform the cleavage in successive steps with one or more intermediate size selection steps. In some embodiments in which targeted fragmentation is used in combination with random or semi-random fragmentation, the random or semi-random nature of the latter may be used for the purpose of achieving Unique Molecular Identifier (UMI) sequences. In some embodiments in which targeted fragmentation is used in combination with random or semi-random fragmentation, the random or semi-random nature of the latter can be used to facilitate sequencing of regions of nucleic acids that are not readily cleaved in a targeted manner, such as long or highly repetitive or substantially similar to other regions in one or more genomes that might otherwise be difficult to enrich by traditional hybrid capture methods.

targeted endonuclease

Targeted endonucleases (eg, CRISPR-associated ribonucleoprotein complexes such as Cas9 or Cpf1, homing nucleases, zinc finger nucleases, TALENs, megaTAL nucleases, arginine nucleases, and/or derivatives thereof ) can be used to selectively cleave and excise targeting moieties of nucleic acid material for the purpose of enriching such targeting moieties for sequencing applications. In some embodiments, targeted endonucleases can be modified, such as with amino acid substitutions, to provide, for example, enhanced thermostability, salt tolerance, and/or pH tolerance or enhanced specificity or alternate PAM site recognition or higher affinity for binding. In other embodiments, the targeted endonuclease may be biotinylated, fused to streptavidin and/or combined with other affinity-based (eg, bait/prey) technologies. In certain embodiments, the targeted endonuclease can have an altered recognition site specificity (eg, a SpCas9 variant with an altered PAM site specificity). In other embodiments, the targeted endonuclease may be catalytically inactive such that cleavage does not occur once bound to the targeting moiety of the nucleic acid material. In some embodiments, the targeted endonuclease is modified to cleave a single strand of the targeting moiety of the nucleic acid material (eg, a nickase variant), thereby creating a gap in the nucleic acid material. CRISPR-based targeted endonucleases are discussed further herein to provide further detailed non-limiting examples of the use of targeted endonucleases. We note that the nomenclature surrounding such targeted nucleases is still in flux. For purposes herein, we use the term "CRISPER-based" to generally refer to an endonuclease that includes a nucleic acid sequence, the sequence of which can be modified to redefine the nucleic acid sequence to be cleaved. Cas9 and CPF1 are examples of such targeted endonucleases currently in use, but there appear to be more such enzymes in different places in nature, and different variants of such targeted and easily regulated nucleases Availability of the body is expected to grow rapidly in the next few years. For example, Cas12a, Cas13, CasX and others are contemplated for use in various embodiments. Similarly, various engineered variants of these enzymes that enhance or alter their properties are becoming available. Herein, we expressly contemplate the use of substantially functionally similar targeted endonucleases not expressly described or discovered herein to achieve similar purposes as disclosed herein.

restriction endonuclease

It is particularly contemplated that any of a variety of restriction endonucleases (ie, enzymes) can be used to provide nucleic acid material of substantially uniform length and/or to excise targeted regions of nucleic acid material. Generally, restriction enzymes are typically produced by certain bacteria/other prokaryotes and cut at, near or between specific sequences in a given DNA segment.

It will be apparent to those skilled in the art that a restriction enzyme is selected to cut at a particular site, or alternatively, at a site created to create a restriction site for cleavage. In some embodiments, the restriction enzyme is a synthetase. In some embodiments, the restriction enzyme is not a synthetase. In some embodiments, a restriction enzyme as used herein has been modified to introduce one or more changes in the genome of the enzyme itself. In some embodiments, restriction enzymes create double-stranded cleavage between defined sequences in a given portion of DNA.

Although any restriction enzyme (eg, Type I, II, III, and/or IV) may be used according to some embodiments, the following represents a non-limiting list of restriction enzymes that may be used: AluI, ApoI, AspHI, BamHI, BfaI, BsaI, CfrI, DdeI, DpnI, DraI, EcoRI, EcoRII, EcoRV, HaeII, HaeIII, HgaI, HindII, HindIII, HinFI, HPYCH4III, KpnI, MamI, MNL1, MseI, MstI, MstII, NcoI, NdeI, NotI, PacI, PstI, PvuI, PvuII, RcaI, RsaI, SacI, SacII, SalI, Sau3AI, ScaI, SmaI, SpeI, SphI, StuI, TaqI, XbaI, XhoI, XhoII, XmaI, XmaII, and any combination thereof. An extensive but non-exhaustive list of suitable restriction enzymes can be found in publicly available catalogs and on the Internet (eg, available at NewEngland Biolabs, Ipswich, MA, USA). It will be appreciated by those skilled in the art that various enzymes, ribozymes or other nucleic acid modifying enzymes that may be used alone or in combination to target phosphodiester backbone cleavage of nucleic acid molecules that may achieve the same purpose may not be included in the above list or Not yet found on the above list. A variety of nucleic acid modifying enzymes can recognize base modifications (e.g., CpG methylation) that can be used to target further modifications of adjacent nucleic acid sequences that can be cleaved (e.g., by enzymes with lyase activity) (e.g., with for the generation of abasic sites). Thus, substantial sequence specificity of cleavage can be achieved based on recognition of DNA or RNA modifications, and this can be used alone or in combination with targeted endonucleases to achieve targeted nucleic acid fragmentation.

Methods for negative and positive enrichment/selection of nucleic acid material

In some embodiments, provided methods and compositions utilize targeted endonucleases (eg, ribonucleoprotein complexes (CRISPR-associated endonucleases such as Cas9, Cpf1), homing endonucleases Enzymes, zinc finger nucleases, TALENs, arginine nucleases and/or meganucleases (eg, megaTAL nucleases, etc., or combinations thereof) or other technologies capable of site-specific interactions with nucleic acid material to positively enrich the desired the (on-target) nucleic acid molecule. Other embodiments provide methods and such compositions for negatively enriching/selecting desired nucleic acid molecules by removing undesired (eg, off-target) nucleic acid material from a sample. Some embodiments described herein combine positive and negative enrichment schemes. In some embodiments, the provided methods can further comprise ligating at least one SMI and/or adaptor sequence to at least one of the 5' or 3' end of the enriched target region. In some embodiments, analysis may be or include quantification and/or sequencing.

In some embodiments, negative enrichment/selection of target nucleic acid material may be facilitated by removing or destroying non-target or unwanted nucleic acid material. 4 is a schematic diagram illustrating the steps of a method of utilizing CRISPR/Cas9 variants to generate targeted nucleic acid fragments of substantially known/selected lengths according to embodiments of the present technology. Use of CRISPR/Cas9 ribonucleoprotein complexes, optionally with enhanced thermostability and/or CRISPR/Cas9 engineered to remain bound to dsDNA under suitable conditions (eg, until removed, enzymatically displaced, etc.) The ribonucleoprotein complex, panel A shows gRNA-promoted binding of variant Cas9 to the targeted DNA site as described above. In one embodiment, and after cleavage and while Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment, the sample can be treated with an exonuclease to hydrolyze the exposed 3' or 5' of the DNA Terminal exposed phosphodiester bonds ( Panel B ). During exonuclease treatment, unwanted or non-targeted DNA will be destroyed by enzymatic activity, leaving only exonuclease resistant target dsDNA fragments. As shown in Figure 4, the bound ribonucleoprotein complex can provide exonuclease protection. Following negative enrichment/selection of target DNA fragments by exonuclease disruption of non-targeted DNA, Cas9 dissociates from DNA and releases blunt-ended double-stranded target DNA fragments of known length, as shown in Panel C. In some embodiments, the method may further comprise the step of incorporating a positive enrichment/selection scheme, such as the use of size selection ( panel D ). In some embodiments, fragments enriched for desired and/or predicted target sizes can be further filtered out genomic fragments that remain undigested and/or protected by off-target Cas9 binding. Optionally, as depicted in Figure E , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing. For example, the blunt end of the target fragment can be directly ligated to a blunt end adaptor. If desired in a particular application, aspects of ligating the adaptor to the cleaved double-stranded nucleic acid material can include end repair and 3'-dA tailing of the fragments. In other embodiments, further processing of fragments to generate ligatable ends of suitable fragments may comprise any of a variety of forms or steps of forming ligatable ends having, for example, blunt ends, A-3 'overhangs, "sticky" ends including one nucleotide 3' overhang, two nucleotide 3' overhangs, three nucleotide 3' overhangs, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides 3' overhang, one nucleotide 5' overhang, two nucleotides 5' Overhangs, three nucleotide 5' overhangs, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide 5' overhangs, etc. The 5' base of the attachment site can be phosphorylated, and the 3' base can have a hydroxyl group, or can be dephosphorylated or dehydrated, alone or in combination, or further chemically modified to facilitate enhanced attachment of one strand to prevent Linking of a chain, optionally, until a later point in time.

In another embodiment, positive enrichment/selection of target nucleic acid material using CRISPR/Cas can be facilitated by affinity-based enrichment of target nucleic acid material. 5 is a schematic diagram illustrating the steps of a method of utilizing CRISPR/Cas9 variants to generate targeted nucleic acid fragments of substantially known/selected lengths according to another embodiment of the present technology. Panel A shows the use of a CRISPR/Cas9 ribonucleoprotein complex, optionally further engineered to retain strong DNA binding under suitable conditions (as described above), wherein the ribonucleoprotein complex includes a capture tag ( such as biotin). The capture label can be bound to a gRNA (eg, crRNA, tracrRNA) or Cas9 protein. Thus, the ribonucleoprotein complex provides an affinity tag for the subsequent pull-down step.

Guide RNA (gRNA)-facilitated binding of the variant Cas9 ribonucleoprotein complex presenting the capture tag is followed by cleavage of double-stranded target DNA. After cleavage and while Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragments, the reaction mixture is contacted with a functionalized surface to which one or more extraction moieties are bound. Extraction moieties provided are capable of binding to capture labels (eg, streptavidin beads, where the capture labels are biotin) for immobilization and separation of capture label bearing molecules. In particular, the extraction moiety may be any member of a binding pair, such as biotin/streptavidin or hapten/antibody or complementary nucleic acid sequence (DNA/DNA pair, DNA/RNA pair, RNA/RNA pair, LNA/DNA pair equal). In the illustrated embodiment, a capture tag attached to a CRISPR/Cas9 ribonucleoprotein complex bound to a (cleaved) target dsDNA fragment is captured by its binding pair (eg, an extraction moiety) that is attached to a Separate fractions (eg, such as magnetically attracted particles or large particles that can be precipitated by centrifugation). Thus, the capture label can be any type of molecule/moiety that allows affinity separation of nucleic acids associated with the capture label (eg, bound by Cas9) from nucleic acids lacking the association of the capture label. An example of a capture label is biotin, which allows affinity separation by binding to streptavidin or oligonucleotides attached or attachable to a solid phase, which in turn Complementary oligonucleotides bind to allow affinity separation. Undesirable or non-targeted nucleic acid material can remain free in solution. Beneficially, free/unbound nucleic acid material without or associated with any capture label can be efficiently removed/separated from the desired target nucleic acid material. In further embodiments, the functionalized surface can be cleaned to remove residual by-products or other contaminants.

Using the affinity-based enrichment scheme shown in Figure 5, the abundance of undesired or non-targeted nucleic acid material can be significantly reduced. Collection of the desired/target nucleic acid fragments can be accomplished in any manner suitable for the application. As a specific example, in some embodiments, collection of the desired nucleic acid material can be accomplished by one or more of the following: defunctionalization by size filtration, magnetic methods, charge methods, centrifugal density methods, or any other method surface, or if a column-based purification method or similar method is used, collect the eluted fraction, or by any other purification practice commonly understood by those skilled in the art.

In some embodiments, an affinity-based positive enrichment step can be combined with a negative enrichment step or can be used in conjunction with a negative enrichment step. For example, after cleavage and while Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment (before or after the affinity-based enrichment step), the sample can be treated with an exonuclease to disrupt the Any unwanted nucleic acid material or contaminants. Following an affinity-based enrichment step and an optional negative exonuclease clean-up step shown in panels A and B , Cas9 dissociates from the DNA to release blunt-ended double-stranded target DNA fragments of known length ( Panel D ). Optionally, the enrichment step described above can be combined with a size-based enrichment step as described above ( Figure E ), and in some embodiments, the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, Sequencing such as described above ( panel F ).

6 is a schematic diagram illustrating the steps of a method for negative enrichment/selection of target nucleic acid material according to another embodiment of the present technology. For example, the enrichment of target double-stranded nucleic acid material can be facilitated by removing or destroying non-target or undesired nucleic acid material. Figure 6 shows an example of using a catalytically inactive variant of Cas9 to generate an enrichment of targeted nucleic acid fragments of substantially known/selected length. Using a catalytically inactive Cas9 ribonucleoprotein complex engineered to target and selectively bind to double-stranded DNA, the gRNA facilitates the binding of a pair of catalytically inactive Cas9 variants to flanking targeted DNA regions ( Figure A ). After binding, the sample can be treated with one or more exonucleases to hydrolyze the exposed phosphodiester bonds of the exposed 3' or 5' ends of the DNA. Catalytically inactive variants of Cas9 do not cleave target DNA, but provide exonuclease resistance such that exonuclease activity cleaves every nucleotide base until blocked by bound Cas9 complexes. Thus, exonuclease treatment destroys all non-targeted nucleic acid material in the sample, with exposed ends leaving fragments protected by pairs of catalytically inactive Cas9. In certain embodiments, mixtures of endonucleases and exonucleases can be used to destroy undesired nucleic acid material. For example, endonucleases (eg, site-specific restriction enzymes) can be used to generate multiple exposed 5' and 3' ends to allow for the enzymatic activity of the exonuclease.

After negative/enrichment selection of target DNA fragments by exonuclease destruction of all non-targeted DNA (Panel B), catalytically inactive Cas9 dissociates from DNA, releasing double-stranded target DNA of known length fragment, as shown in Figure C. As discussed above, additional size selection steps can be performed for further enrichment of target double-stranded DNA fragments ( panel D ). Optionally, the enriched DNA fragments can be polished, blunted or tailed to form suitable ligable ends, and subsequently ligated to adaptors for nucleic acid interrogation, such as sequencing ( Figure E ).

In another example depicted in Figure 7, both negative and positive enrichment protocols can be implemented using catalytically inactive variants of Cas9. Panel A shows the use of a catalytically inactive variant of Cas9 in a ribonucleoprotein complex engineered to remain bound to DNA under appropriate conditions, and wherein the ribonucleoprotein complex includes Capture label (eg, on guide RNA or tethered to Cas9 protein). Catalytically inactive variant Cas9 ribonucleoprotein complexes with capture-labeled guide RNA (gRNA)-promoted binding are followed by the addition of exonuclease to the sample to hydrolyze the exposed 3' or 5' ends of the DNA phosphodiester bond. Catalytically inactive variants of Cas9 do not cleave target DNA, but provide exonuclease resistance such that exonuclease activity cleaves every nucleotide base until blocked by bound Cas9 complexes. After negative/enriched selection of target DNA fragments by exonuclease destruction of all non-targeted DNA, and while catalytically inactive Cas9 remains bound, stepwise additions capable of binding to the ribonucleoprotein complex (when its A functionalized surface (e.g., a functionalized surface having one or more extraction moieties bound thereto) that retains the capture label associated with the target nucleic acid) can be immobilized and/or separated carrying the capture label and/or associated with the capture The associated molecules are labeled with undesired nucleic acid material retained in the sample ( Panel B ). In some embodiments, provided methods allow for the removal of all or substantially all unwanted nucleic acid material in a sample, or substantially reduce their abundance. Collection of the desired target nucleic acid material can be accomplished in any manner suitable for the application. As a specific example, in some embodiments, collecting the desired target nucleic acid fragments can be accomplished by one or more of the following: functional removal by size filtration, magnetic methods, charge methods, centrifugal density methods, or any other method The eluted surface is collected, or if a column-based purification method or similar method is used, the eluted fraction is collected, or by any other commonly understood purification practice.

Following an affinity-based enrichment step, and as depicted in Figure D , Cas9 dissociates from DNA and releases double-stranded target DNA fragments of known length. Panel E depicts an optional further processing step for positive enrichment/selection of target DNA fragments by size selection. Optionally, as depicted in Figure F , the enriched DNA fragments can be ligated to adaptors for nucleic acid interrogation, such as sequencing.

In some embodiments, a combination of catalytically active and catalytically inactive CRISPR/Cas complexes can be used to positively enrich for fragments comprising target double-stranded nucleic acid regions. Referring to Figure 8, both catalytically active and catalytically inactive Cas9 ribonucleoprotein complexes can target desired nucleic acid regions (eg, specific genomic loci) in a sample in a sequence-dependent manner. Catalytically active Cas9 ribonucleoprotein complexes are directed to flanking regions of the target DNA region and are used to cleave the target double-stranded DNA to release blunt-ended double-stranded target DNA fragments of known length. One or more catalytically inactive ribonucleoprotein complexes with a capture label (eg, biotin) are directed to the region of the target sequence between the two site-selected cleavage sites. After cleavage of target DNA to release DNA fragments, the addition of functionalized surfaces capable of binding capture labels associated with catalytically inactive ribonucleoprotein complexes can facilitate positive enrichment/selection of target fragments. It will be appreciated that many other forms of targeted nucleic acid fragmentation, such as those described above, can be substituted for the active Cas9 ribonucleoprotein complex in this example.

In some embodiments, a positive enrichment/selection step can be taken to enrich for target sequences from samples where the nucleic acid material has been fragmented (eg, mechanically sheared or from a cell-free DNA sample (eg, from a liquid) biopsy)). Figures 9A and 9B are conceptual illustrations of method steps for positive enrichment/selection of target nucleic acid fragments using catalytically inactive variants of the Cas9 ribonucleoprotein complex with capture tags as described above. Fragmented double-stranded DNA fragments (eg, mechanical shearing, acoustic fragmentation, cell-free DNA, etc.) in a sample can be directed via targeting of one or more catalytically inactive Cas9 ribonucleoprotein complexes in solution. Binding was positively enriched/selected (Figure 9A).

In some embodiments, a method can comprise the use of two or more capture labels (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), which can be used with for differentially labeling multiple Cas9 ribonucleoprotein complexes. For example, a sample can be enriched for multiple target nucleic acid samples simultaneously. While in some embodiments it is expected that all Cas9 complexes carry the same capture label (eg, biotin) so that all targeted sequences can be pulled down (affinity purified) together in a single sample, in In other embodiments, separation of differently targeted sequences can be facilitated by conjugating substantially unique capture labels to Cas9 complexes targeting different regions. In some embodiments, the at least two capture labels used in the method are different from each other (eg, small molecules and peptides). In some embodiments, the inclusion of two or more different capture labels allows the use of both positive enrichment/selection and negative enrichment/selection. The inclusion of two or more capture labels may be helpful, especially in situations where it is desired to physically separate nucleic acid fragments comprising different target sequences for subsequent nucleic acid interrogation (eg, sequencing).

The reaction mixture is contacted with a functionalized surface to which one or more extraction moieties are bound. The provided extraction fractions are capable of binding to capture labels (eg, streptavidin beads, where the capture labels are biotin) for immobilization and isolation of capture label-bearing molecules (Figure 9B).

In some embodiments, it is desirable to enrich or isolate target nucleic acid material from the sample when the sample contains fragments of different sizes, which are smaller and which might otherwise be lost during processing steps (eg, DS process steps) fragment size. Figure 10 is a schematic diagram showing the method steps for positive enrichment/selection of target nucleic acid fragments using catalytically inactive variants of the Cas9 ribonucleoprotein complex with capture tags. Panel A shows multiple fragmented double-stranded DNA fragments of different sizes in the sample, including molecule 2, which is too small to be reliably enriched by size selection or affinity-based methods. In this example, adapters (eg, sequencing adapters) can be ligated/attached to fragment ends using known sequencing library preparation procedures. In this way, certain small nucleic acid fragments are extended by flanking adaptor molecules. Positive enrichment of targeted fragments from solution can be performed as described above with respect to Figures 9A and 9B. For example, Panel B of Figure 10 shows the ligation of adaptors to the 5' and 3' ends of the molecules in the sample, thereby making such DNA fragments longer in length. Panel C shows a positive enrichment/selection step for molecule 2 by targeting directed binding of the catalytically inactive Cas9 ribonucleoprotein complex with a capture label in solution, followed by affinity purification.

11 is a schematic diagram illustrating the steps of a method for enriching targeted nucleic acid material using a negative enrichment scheme ( panel A ) and a positive enrichment scheme ( panel B ) in accordance with an embodiment of the present technology. Panel A shows the ligation of hairpin adaptors to the 5' and 3' ends of a double-stranded target DNA molecule to generate adaptor-nucleic acid complexes without exposed ends. Adapter-nucleic acid complexes are treated with exonuclease in a negative enrichment/selection protocol to eliminate fragments of nucleic acid material and adapters with unprotected 5' and 3' ends (eg, no 4 linked phosphodi Ester-linked adaptor-nucleic acid complexes, unligated DNA, single-stranded nucleic acid material, free adaptors, etc.), as shown on the right side of panel B.

As shown in Figure 11, a hairpin adaptor can include a cleavable moiety in the linker moiety, such as a uracil group, or any other enzymatically, chemically or optoelectronically cleavable group. When using uracil DNA glycosylase (UDG) and an enzyme with abasic site DNA lyase activity (such as endonuclease VIII or formamidopyrimidine [fapy]-DNA glycosylase (FPG)) or Cleavage at uracil can convert hairpin adaptors to include Y-shapes suitable for polymer clone formation (bridging amplification) and certain sequencing modes when processed by a combination of commercial premixed combinations (e.g., USER ^™ enzymes). linker.

Exonuclease-resistant adaptor-nucleic acid complexes can be further enriched by size selection or by target sequences (eg, CRISPR/Cas9 pulldown) (Figure 11, left panel B ). In another embodiment, hairpin adaptors with capture tags (as shown in Figure 12) can be used, which are directly suitable for affinity-based enrichment using functionalized surfaces with exposed extraction moieties.

In the embodiment described in Figure 11 after negative enrichment of target nucleic acid fragments ligated to hairpin adaptors, an additional positive enrichment step can be performed. For example, Figure 13 is a schematic diagram showing the method steps for positive enrichment of adaptor-target nucleic acid complexes using hairpin adaptors ( Panel A ) followed by rolling circle amplification ( Panels B and C ). The rolling circle amplification step can be used to (1) provide a substantially 1:1 ratio of first-strand amplicons to second-strand amplicons, and (2) prevent stranding prior to labeling and/or during library clean-up steps dissociate. Long-molecule sequencing platforms may be suitable for direct sequencing of rolling circle amplicons (panel C); however, for short-read sequencing platforms, one can (1) enzymatically cleave cleavage sites including cleavage sites (e.g., restriction endonuclease recognition sites) dots) to generate approximately uniform ratios of first-strand and second-strand amplicons (left side of panel D ), or (2) use PCR amplification to generate multiple components including substantially the same Proportion of short amplicons of the first and second sequences (right side of panel D ).

Figure 14 is a diagram illustrating a method for generating targeted nucleic acid fragments of known/selected lengths with different 5' and 3' ligatable ends using site-directed binding and cleavage of CRISPR/Cpf1 Schematic of the steps. In various embodiments, the 5' and 3' ligatable ends include single-stranded overhang regions of known nucleotide length and sequence. Cpf1 is a targeted endonuclease that recognizes a T-rich PAM at the targeted 5' end and performs staggered cleavage in double-stranded DNA target sequences. For example, a variant of Cpf1 cleaves 19 bp after PAM on the sense strand and 23 bp on the antisense strand, as shown in FIG. 14 . Panel A shows gRNA-promoted binding of Cpf1 at targeted DNA sites. Cpf1-directed cleavage generates staggered cleavage, providing 4 (depicted) or 5 nucleotide overhangs (eg, "sticky ends"). Site-directed Cpf1 cleavage flanking the target DNA sequence generates double-stranded target DNA fragments of known length (eg, which can be further and optionally enriched by size selection) with sticky ends 1 5' to the fragment end and sticky end 2 at the 3' end of the fragment ( panel B ). Panel B further shows ligation of adaptor 1 at the 5' end of the fragment, and ligation of adaptor 2 at the 3' end of the fragment, wherein adaptor 1 and adaptor 2 comprise at least part of the cohesive ends 1 and 2 on the fragment, respectively complementary overhang sequences.

By design, the sequence of sticky end 1 (the overhang at the 5' end of the targeted fragment) is known. Likewise, the sequence of sticky end 2 (the overhang at the 3' end of the targeted fragment) is known. Specific adaptors can be synthesized that include substantially complementary sequences such that the fragments can be ligated to the adaptors at both ends. In one embodiment, the adaptors can be the same type of adaptors (eg, adaptors including Y-shaped, U-shaped, barcoded adaptors, etc.). In another embodiment, the adaptors can be different (eg, adaptor 1 can comprise a Y shape and adaptor 2 can comprise a U shape). Other unique features may include different primer sites for amplification, different types or positions of barcodes or other unique molecular identifiers, adapters including capture tags and adapters without capture tags, some adapters may include Fluorescent labels, etc. In some applications, there are distinct advantages to designing specific adaptors to be located at the 5' or 3' end of the fragment. The specificity of the essentially unique sticky ends on the targeted fragments facilitates these types of applications. In addition, positive selection of successfully cleaved and adaptor-ligated target fragments ensures that only target-enriched nucleic acid regions are amplified and sequenced.

In some embodiments, substantially unique sticky ends generated by Cpf1 cleavage can be used for additional positive enrichment protocols. For example, FIG. 15 is a diagram illustrating steps of a method for affinity-based enrichment of target DNA fragments comprising sticky ends (eg, such as those generated in the method of FIG. 14 ), according to embodiments of the present technology. Schematic. Panel A shows the stepwise addition of functionalized surfaces capable of binding sticky ends associated with target DNA fragments cleaved in solution. For example, a functionalized surface may have bound thereto one or more extraction moieties that are suitable as binding pairs with one or more targeted DNA overhang sequences. The provided extraction moiety can be, for example, a synthetic oligonucleotide having a predefined or known oligonucleotide sequence that is at least partially complementary to the resulting cohesive ends of the Cpf1-cleaved target sequence. Oligonucleotides can include DNA, RNA, or LNA sequences capable of binding to capture labels (eg, sticky ends) for immobilization and isolation of targets including sticky ends. Once bound to the functionalized surface, affinity interactions facilitate pull-down (eg, affinity purification) of the desired double-stranded DNA fragments, while discarding non-targeted fragments, as shown in panel B.

16 is a diagram illustrating a method for affinity-based enrichment of target DNA fragments comprising sticky ends (eg, such as those generated in the method of FIG. 14 ) in accordance with another embodiment of the present technology Schematic of the steps. Panel A shows the stepwise addition of capture-labeled oligonucleotides having a predefined or known oligonucleotide sequence that is at least partially associated with in solution A portion of the cleaved target DNA fragment associated cohesive ends is complementary. In certain examples, oligonucleotide strands can be synthesized in the 3' to 5' direction, such as by phosphoramidite methods (eg, on controlled pore glass (CPG) fragments, etc.), and chemical moieties can be The nucleotides are linked (eg, covalently, non-covalently, ionic, or other linking chemistry) to the 5' end after synthesis, or as part of the synthesis of oligonucleotides, such as by The incorporation of a non-standard phosphoramidite molecule near the 5' end or at an internal position in the oligonucleotide is ligated.

As shown in Panel B , further addition of a functionalized surface capable of binding a capture label facilitates pull-down (eg, affinity purification) of the desired double-stranded DNA fragments, while discarding non-targeted fragments.

Referring to Figures 15 and 16 together, and in a subsequent step (not shown), elution of the targeted fragment may be performed by release from the extraction fraction. In some non-limiting examples, the cleavable moiety can be bound near the binding end of the oligonucleotide extraction moiety. In another example, the temperature or other conditions can be altered to cause denaturation of the short capture label/extract binding while maintaining the double-stranded nature of the target nucleic acid fragment. In yet another embodiment, a hairpin adaptor can be used at the second cohesive end of the target fragment to tether the double strands together during elution and further processing. In various embodiments, after the enrichment step, as described herein, the sticky ends may be polished, trimmed, or biocomputationally filtered to avoid false recombination errors.

17 is a schematic diagram showing the steps of a method for targeted fragment enrichment of nucleic acid material of known length and having different 5' and 3' ligatable ends using Cas9 nickase, according to an embodiment of the present technology, The ligable ends include long single-stranded overhangs of known nucleotide length and sequence. Panel A shows gRNA-targeted binding of paired Cas9 nickases in targeted DNA regions. Double-strand breaks can be introduced by excising target DNA regions using paired nickases, and when paired Cas9 nickases are used, long overhangs are created on each cleaved end (sticky ends 1 and 2), As shown in Figure B. Thus, in contrast to cleavage with catalytically active Cas9 that produces blunt ends, strategic pairing of Cas9 nickases can provide staggered single-stranded cleavage on opposing DNA strands to generate long overhangs, as depicted in panel B. As described above with respect to Figure 15, the stepwise addition of a functionalized surface capable of binding the long sticky ends associated with cleaved target DNA fragments in solution (eg, sticky end 1) provides targeted DNA fragments in solution Positive enrichment step. For example, the extraction moiety may be an oligonucleotide having a predefined or known oligonucleotide sequence that is substantially complementary to the predefined or known sequence of the long sticky ends of the fragment. Once bound to the functionalized surface, affinity interactions facilitate pull-down (eg, affinity purification) of the desired double-stranded DNA fragments, while discarding non-targeted fragments, as shown in panel D.

Figure 17, Panel E shows a variant of the positive enrichment step involving the addition and annealing of capture-labeled oligonucleotides with predefined or known oligonucleotide sequences, which The oligonucleotide sequence is at least partially complementary to a portion of the long sticky end (eg, sticky end 1) associated with the cleaved target DNA fragment in solution. Panel F shows the annealing of a second oligo strand that is at least partially complementary to a portion of the capture-labeled oligonucleotide. Enzymatic extension of the second oligo strand and ligation to the template DNA fragment generates the adaptor-target DNA complex. As shown, the first and second oligonucleotide strands comprise single-stranded moieties such that the resulting adaptor complexes comprise asymmetry for DS processing. Furthermore, the first oligonucleotide strand may comprise a degenerate or semi-degenerate SMI sequence, such that when the second oligonucleotide strand is extended, the first oligonucleotide strand acts as a template strand and the SMI sequence is Made into double strands. A further step may involve the introduction of a functionalized surface (not shown) capable of binding a capture label to facilitate pull-down (e.g., affinity purification) of the desired adaptor-dsDNA complex, while discarding non-targeted fragments .

Various aspects of the present technology include methods for negatively enriching nucleic acid regions by providing exonuclease and endonuclease resistance via protein binding. In one example, as shown in Figure 18, site-selected proteins that bind to target DNA can be used to provide exonuclease and endonuclease resistance. As shown, the target nucleic acid enrichment protocol uses the catalytically inactive Cas9 ribonucleoprotein complex to protect targeted genomic regions. With gRNA, Cas9 can be targeted to the desired sequence in the sample. One or more catalytically inactive ribonucleoprotein complexes with one or more capture tags can be positioned in close proximity and/or adjacent positions to protect regions of genomic DNA from enzymatic digestion. In some embodiments, as shown, ribonuclease complexes can be engineered to direct other protein complex structures to target DNA regions. Exonuclease resistance is provided when the protein complex structure covers the target DNA region. After treatment with an exonuclease or a combination of an endonuclease and an exonuclease, affinity purification of the protein complex (eg, by capture tags bound to functionalized surfaces, antibody pulldown, etc.) removes target DNA fragments Separated from other unwanted nucleic acid material or unbound proteins in solution. The target nucleic acid fragment can then be released from the ribonucleotide complex binding.

Nucleic acid libraries and methods for making and using nucleic acid libraries

In some embodiments, the provided methods can comprise the steps of: providing a nucleic acid material, directing a plurality of targeted catalytically inactive endonucleases (eg, ribonucleoprotein complexes) to cells distributed along the nucleic acid material multiple regions to generate nucleic acid libraries that can be interrogated by selective probes at any time.

19A and 19B are conceptual illustrations of DNA libraries and reagents prepared according to embodiments of the present technology that can be used as tools to selectively interrogate relevant DNA regions. Uniquely labeled catalytically inactive Cas9 targets multiple (eg, spaced) regions of isolated/unfragmented genomic DNA (or other large DNA fragments) (FIG. 19A). Each catalytically inactive Cas9 ribonucleoprotein includes a known oligonucleotide tag with a known sequence (eg, a code sequence) and binds to a predesigned region of the genome. As shown schematically in Figure 19A, multiple inactive Cas9 ribonucleoprotein complexes (e.g., ^iCas9A , ^iCas9B , ^iCas9C , ^iCas9N ) are directed by gRNAs to bind distributed throughout the genome region (e.g., large selected regions, entire genomes, etc.) of the genomic loci (Site ^A , Spot ^B , Spot ^C , Spot ^N ). Each iCas9 complex includes an oligonucleotide tag including an oligonucleotide code sequence (AAAAAAA), where "A" is any nucleotide (unmodified or modified). The leading position of the nucleotide includes a substantially unique code that can be recorded and then looked up in a look-up table.

When a specific target sequence or smaller region needs to be interrogated (eg, sequenced), the library can be probed with specially designed capture probes designed to pull down the desired region. A method of fragmentation can be used to fragment genomic DNA into various sizes (eg, restriction enzyme digestion, mechanical shearing, etc.). Since each iCas9 complex includes a substantially unique oligonucleotide tag that is computationally associated with a DNA locus, the user can incrementally add one or more probes that include codes corresponding to regions of the genome of interest The complement of the sequence (eg, the reverse code sequence). For example, and as shown in Figure 19B, an inverse code sequence is a nucleotide sequence that is substantially complementary to the associated code sequence. For example, to extract the region that includes site ^A , the user looks up the code sequence associated with the iCas9A complex that binds to site ^A (AAAAAAA). This can then be achieved by introducing an Partially functionalized surfaces (eg streptavidin, where biotin is the capture label) to functionally select and enrich regions of interest.

In various embodiments, a nucleic acid library can be used as a resource for interrogation of several probes. In addition, several libraries can be prepared that are pre-bound with multiple CRISPR/Cas site-directed complexes. In addition, some libraries can be pre-fragmented or cleaved using mechanical shearing, endonuclease cleavage (using one or more restriction endonucleases). When the desired target region is excised (eg, by targeted endonuclease digestion (eg, CRISPR/Cas, restriction enzymes, etc.), the length of the target fragment will be known, and after pulldown using the probe , target fragments can be further enriched by size selection.

another method

Some aspects of the present technology are applicable to long sequence sequencing technologies, such as direct digital sequencing (DDS) platforms. In some embodiments, it is desirable to enrich for relevant target sequences for DDS. In such embodiments, amplification-free enrichment of the target sequence is desired. Furthermore, it is further desirable to generate double-stranded sequencing data on such platforms.

20 illustrates steps of a method for affinity-based enrichment and sequencing of target DNA fragments for use with a direct digital sequencing method in accordance with an embodiment of the present technology. Panel A shows selected adaptor ligations to target DNA fragments including cohesive ends (eg, such as those generated in the methods of FIG. 14 or FIG. 17 ). Panel A further shows ligation of adaptor 1 at the 5' end of the fragment, and ligation of adaptor 2 at the 3' end of the fragment, wherein adaptor 1 and adaptor 2 comprise at least part of the cohesive ends 1 and 2 on the fragment, respectively complementary overhang sequences. Adaptor 1 has a Y shape and includes 5' and 3' single-stranded arms with different labels (A and B) including different properties. Adaptor 2 is a hairpin adaptor.

Panel B shows steps in a direct digital sequencing method, where label A is configured to bind to a functional surface. Label B provides physical properties (eg, charge, magnetism, etc.) such that application of an electric or magnetic field results in denaturation of the first and second strands of the double-stranded adaptor-DNA complex, followed by electrical stretching of the DNA fragments. The first and second strands remain tethered by hairpin adaptors, so that sequence information from the enriched/targeted strands provides double-stranded sequence information for error correction and other nucleic acid interrogations (eg, assessment of DNA damage, etc.). For example, the sequence generated from the first strand can be compared to the sequence generated from the second strand for error correction, or in another example, to determine the site and characteristics of DNA damage. In some embodiments, the enriched targeted genomic region may have a length between about 1 and 1,000,000 bases. For example, in some embodiments, and when denatured and sequenced, the length of the enriched nucleic acid fragments can be at least 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 15; 20; 25; 30; 35; 40; 50; 60; 70; 80; 90; 100; 120; 150; 200; 300; 400; 4000; 5000; 6000; 7000; 8000; 9000; 10,000; 15,000; 20,000; 30,000; In some embodiments, fragments can be up to 60,000; 70,000; 80,000; 90,000; 100,000; 120,000; 150,000; 200,000;

21 illustrates steps of an affinity-based enrichment method for sequencing target DNA fragments using a DDS method according to another embodiment of the present technology. Panel A shows affinity-based enrichment of target DNA fragments including cohesive ends (eg, such as those generated in the methods of FIG. 14 or FIG. 17 ). As shown, the hairpin adaptor has been ligated to the 3' end of the double-stranded DNA fragment in a sequence-dependent manner. Target DNA molecules can flow over a functionalized surface (eg, with bound oligonucleotides) capable of binding sticky ends associated with cleaved target DNA fragments. Additionally, a second oligonucleotide strand comprising label B and at least partially complementary to a portion of the bound oligonucleotide is added to the solution. Annealing and ligation of the adaptor/DNA fragment components provides adaptor-target double-stranded DNA complexes that bind to a surface suitable for direct digital sequencing ( Panel B ). The application of electric or magnetic fields for the sequencing step and the electrical stretching of the adaptor-DNA complex can be performed as described, for example, in FIG. 20 .

Reagents and Methods

adaptor type

While most examples in this disclosure depict Y-shaped or circular adaptors, according to various embodiments, any known adaptor structure may be used, such as in WO2017/100441 (which is incorporated herein by reference in its entirety) those described in. For example, various adaptor shapes including bubbles (eg, non-complementary interior regions) are also further considered.

separate

As described herein, various methods comprise at least one separation step. It is specifically contemplated that any of the various separation steps may be included in various embodiments. For example, in some embodiments, separation may be or include physical separation, size separation, magnetic separation, solubility separation, charge separation, hydrophobic separation, polar separation, electrophoretic mobility separation, density separation, chemical elution separation, SBIR Bead separation, etc. For example, a physical group can have magnetic properties, charge properties, or insolubility properties. In an embodiment, when the physical group has magnetic properties and a magnetic field is applied, the associated adaptor nucleic acid sequence comprising the physical group is separated from the adaptor nucleic acid sequence that does not comprise the physical group. In another embodiment, when the physical group has a charge characteristic and an electric field is applied, the associated adaptor nucleic acid sequence comprising the physical group is separated from the adaptor nucleic acid sequence that does not comprise the physical group. In an embodiment, when the physical group has insoluble properties and the adaptor nucleic acid sequence is contained in a solution in which the physical group is insoluble, the adaptor nucleic acid sequence comprising the physical group never contains the physical group remaining in solution. The adaptor nucleic acid sequence precipitates out.

Any of a variety of physical separation methods may be included in various embodiments. As a specific example, a non-limiting set of methods includes: size selective filtration, density centrifugation, HPLC separation, gel filtration separation, FPLC separation, density gradient centrifugation, gel chromatography, and the like.

Any of various magnetic separation methods may be included in various embodiments. Typically, a magnetic separation method will involve the inclusion or addition of one or more physical groups having magnetic properties such that when a magnetic field is applied, molecules comprising such physical groups are separated from molecules not comprising such physical groups. As specific examples, physical groups exhibiting magnetic properties include, but are not limited to, ferromagnetic materials, such as iron, nickel, cobalt, dysprosium, gadolinium, and alloys thereof. Commonly used paramagnetic beads for chemical and biochemical separations embed such materials in a surface that reduces chemical interactions of the material with manipulated chemicals, such as polystyrene, which can target the aforementioned affinity Features are functionalized.

capture marker

As described herein, in some embodiments, capture labels may be present in any of a variety of configurations along oligonucleotide probes, adaptors, ribonucleotide sequences, ribonucleoprotein complexes, and the like on protein. In some embodiments, a capture label can be incorporated or attached to an oligonucleotide strand in a region 5' of the sequence. In some embodiments, the capture label may be present somewhere in the middle of the oligonucleotide chain (ie, not at the 5' or 3' end of the oligonucleotide). In embodiments comprising two or more capture labels, each capture label may be present at a different location along the oligonucleotide.

In some embodiments, the capture label is selected from the group consisting of biotin, biotin deoxythymidine dT, biotin NHS, biotin TEG, biotin-6-aminoallyl-2'-deoxyuridine-S '-triphosphate, biotin-16-aminoallyl-2-deoxycytidine-5'-triphosphate, biotin-16-aminoallylcytidine-5'-triphosphate, N4-bio Biotin-OBEA-2'-deoxycytidine-5'-triphosphate, Biotin-16-aminoallyluridine-5'-triphosphate, Biotin-16-7-deaza-7-amino Allyl-2'-deoxyguanosine-5'-triphosphate, 5'-biotin-G-monophosphate, 5'-biotin-A-monophosphate, 5'-biotin-dG- Monophosphate, 5'-biotin-dA-monophosphate, desthiobiotin NHS, desthiobiotin-6-aminoallyl-2'-deoxycytidine-5'-triphosphate, digoxigenin NHS, DNP, TEG, thiols, colicin E2, Im2, glutathione, glutathione-s-transferase (GST), nickel, polyhistidine, FLAG tag, myc-tag, etc. In some embodiments, capture labels include, but are not limited to, biotin, avidin, streptavidin, haptens recognized by antibodies, specific nucleic acid sequences, and/or magnetically attractive particles. In some embodiments, one or more chemical modifications of nucleic acid molecules (eg, Acridite-modified and many others, some of which are described elsewhere in this application) can be used as capture labels.

Extract part

The extraction moiety can be a physical binding partner or pair targeted to a capture label, and refers to an detachable moiety or any type of molecule that allows capture-labeled nucleic acid or capture-labeled molecules (e.g., oligonucleotides) Affinity separation of nucleic acids bound to acids, proteins, ribonucleoprotein complexes, etc. from nucleic acids lacking capture labels. The extraction moiety can be attached directly or indirectly (eg, by nucleic acid, by antibody, by adaptor, etc.) to a substrate (such as a solid surface). In some embodiments, the extraction moiety is selected from the group consisting of small molecules, nucleic acids, peptides, antibodies, or any uniquely bindable moiety. The extraction moiety can be or can be linked to a solid phase or other surface for forming a functionalized surface. In some embodiments, the extraction moiety is a sequence of nucleotides attached to a surface (eg, a solid surface, beads, magnetic particles, etc.). In some embodiments wherein the capture label is biotin, the extraction moiety is selected from the group of avidin or streptavidin. Those skilled in the art will understand that any of a variety of affinity binding pairs may be used according to various embodiments.

In certain embodiments, the extraction moiety may be a physical or chemical property that interacts with the targeted capture label. For example, the extraction moiety can be a magnetic field, a charge field, or a liquid solution in which the targeted capture label is insoluble. Such physical or chemical properties can be applied, and the adapter nucleic acid with the capture label can be immobilized in/against the container (surface) or column. Depending on the desired positive enrichment/selection or negative enrichment/selection result, either immobilized molecules (positive enrichment) or non-immobilized molecules (negative enrichment) may be retained for further purification/processing or use.

solid surface

When the affinity partner/extraction moiety is attached to a solid surface or substrate and bound to the capture label, the adaptor nucleic acid sequence comprising the capture label can be separated from the adaptor nucleic acid sequence that does not comprise the affinity label. The solid surface or substrate can be beads, separable particles, magnetic particles, or another immobilized structure.

As described herein, and as will be understood by those skilled in the art, any of a variety of functionalized surfaces may be used according to various embodiments. For example, in some embodiments, the functionalized surface can be or include beads (eg, controlled pore glass beads, macroporous polystyrene beads, etc.). However, those skilled in the art will appreciate that many other chemical moiety/surface pairs can be similarly used to achieve the same purpose. It will be appreciated that the specific functionalized surfaces described herein are by way of example only and that any other suitable immobilization structure capable of being associated with (eg, attached to, bound to, one or more extraction moieties, etc.) one or more extraction moieties may be used or base.

Nucleic acid cleavage

Various aspects of the present technology include enriching nucleic acid material using adaptors, oligonucleotides and capture labels that can incorporate enzymatic cleavage, single-stranded enzymatic cleavage, double Enzymatic cleavage of strands, incorporation of modified nucleic acids followed by enzymatic treatment leading to cleavage of one or both strands, incorporation of photocleavable linkers, incorporation of uracil, incorporation of ribobases, incorporation of 8-oxo Substitute guanine adducts, use restriction endonucleases, use site-directed cleavage enzymes, etc. In other embodiments, endonucleases, such as ribonucleoprotein endonucleases (eg, Cas enzymes such as Cas9 or CPF1), or other programmable endonucleases (eg, homing endonucleases, Zinc finger nucleases, TALENs, meganucleases (eg, megaTAL nucleases, arginine nucleases, etc.), and any combination thereof.

As described herein, various embodiments include the use of one or more endonucleases that recognize unique nucleotide sequences or modifications or other entities that recognize bases or other backbone chemical modifications for use in one or more Specific positions in the strands cut and/or cleave double-stranded nucleic acids (eg, DNA or RNA). Examples include uracil (recognized and can be cleaved by a combination of uracil DNA glycosylase and abasic site cleavage enzymes such as endonuclease VIII or FPG) and ribonucleotides, when these ribonucleotides are combined with During DNA base pairing, ribonucleotides can be recognized and cleaved by RNAseH2. The nucleic acid can be DNA, RNA, or a combination thereof, and optionally, comprises a peptide nucleic acid (PNA) or locked nucleic acid (LNA) or other modified nucleic acid. In some embodiments, cleavage can be performed using one or more restriction endonucleases. In some embodiments, cleavage can be performed using a cleavable linker (eg, uracil desthiobiotin-TEG, ribose cleavage, or other methods). In some embodiments, the cleavable linker may be a photocleavable linker or a chemically cleavable linker that does not require or partially require an enzyme.

One of ordinary skill in the art will understand that various restriction endonucleases (ie, restriction enzymes) that cleave DNA at or near the recognition site (eg, EcoRI, BamHI, XbaI, HindIII, AluI, AvaII, BsaJI, BstNI, DsaV, Fnu4HI, HaeIII, MaeIII, NlaIV, NSil, MspJI, FspEI, NaeI, Bsu36I, NotI, HinF1, Sau3AI, PvuII, SmaI, HgaI, AluI, EcoRV, etc.) may conform to various embodiments of the present technology. Lists of several restriction endonucleases are available in printed and computer readable form and are provided by a number of commercial suppliers (eg, New England Biolabs, Ipswich, MA). A non-limiting list of restriction endonucleases and associated recognition sites can be found at www.neb.com/tools-and-resources/selection-charts/alphabetized-list-of-recognition-specificities.

In some embodiments, modified or non-nucleotides can provide cleavable moieties. For example, uracil bases (as an example, can be cleaved with a combination of UGD and endonuclease VIII or FPG), abasic sites (as an example, can be cleaved with endonuclease VIII), 8 -oxo-guanine (can be cleaved with FPG or OGG1 as an example) and ribonucleotides (in one example, when paired with DNA, can be cleaved with RNAseH2).

connectable end

In some embodiments, adaptor products are generated that have ligable 3' ends suitable for ligation to target double-stranded nucleic acid sequences (eg, for sequencing library preparation). The ligation domains present in each double-stranded adaptor product can be capable of being ligated to a corresponding strand of the double-stranded target nucleic acid sequence. In some embodiments, one of the linking domains comprises a T-overhang, A-overhang, CG-overhang, polynucleotide overhang, blunt end, or another ligable nucleic acid sequence. In some embodiments, the double-stranded 3' linking domain includes blunt ends. In certain embodiments, at least one of the linker domain sequences comprises a modified or non-standard nucleic acid. In some embodiments, the modified nucleotide can be an abasic site, uracil, tetrahydrofuran, 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-A) , 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-G), deoxyinosine, 5'-nitroindole, 5-hydroxymethyl-2'-deoxy Cytidine, isocytosine, 5'-methyl-isocytosine, or isoguanosine. In some embodiments, at least one strand of the linker domain comprises dephosphorylated bases. In some embodiments, at least one of the linking domains comprises a dehydroxylated base. In some embodiments, at least one chain of the linking domain has been chemically modified so as to render it non-ligable (eg, until further action is taken to render the linking domain ligable). In some embodiments, 3' overhangs are obtained by using a polymerase with terminal transferase activity. In one example, Taq polymerase can add a single base pair overhang. In some embodiments, this is "A".

non-standard nucleotides

In some embodiments, the provided template and/or extension chain may comprise one or more non-standard/non-canonical nucleotides. In some embodiments, non-standard nucleotides can be or include uracil, methylated nucleotides, RNA nucleotides, ribonucleotides, 8-oxo-guanine, biotinylated nucleotides, Dethiobiotin nucleotides, thiol-modified nucleotides, acrylate-modified nucleotides, iso-dC, iso-dG, 2'-O-methyl nucleotides, inosine nucleotides locked nucleic acids, peptides Nucleic acids, 5-methyl dC, 5-bromodeoxyuridine, 2,6-diaminopurine, 2-aminopurine nucleotides, abasic nucleotides, 5-nitroindole nucleotides, adenylation Nucleotides, azide nucleotides, digoxigenin nucleotides, I-linkers, 5'hexynyl modified nucleotides, 5-octadiynyl dU, photocleavable spacers, non- Photocleavable spacers, click chemistry compatible modified nucleotides, fluorescent dyes, biotin, furan, BrdU, fluoro-dU, loto-dU, and any combination thereof.

another aspect

According to aspects of the present disclosure, some embodiments provide high quality sequencing information from very small amounts of nucleic acid material. In some embodiments, provided methods and compositions can be combined with up to about 1 picogram (pg); 10 pg; 100 pg; 1 nanogram (ng); 10 ng; 100 ng; , 800ng, 900ng or 1000ng of starting nucleic acid material were used together. In some embodiments, the provided methods and compositions can be used with an input amount of nucleic acid material of up to 1 molecular copy or genomic equivalent, 10 molecular copies or genomic equivalent thereof, 100 Molecular copies or genomic equivalents thereof, 1,000 molecular copies or genomic equivalents thereof, 10,000 molecular copies or genomic equivalents thereof, 100,000 molecular copies or genomic equivalents thereof, or 1,000,000 molecular copies or genomic equivalents thereof. For example, in some embodiments, up to 1,000 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 10 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 pg of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 pg of nucleic acid material is initially provided for a particular sequencing process.

According to other aspects of the present technology, some of the provided methods can be used to sequence any of a variety of suboptimal (eg, damaged or degraded) samples of nucleic acid material. For example, in some embodiments, at least some of the nucleic acid material is damaged. In some embodiments, the damage is or includes oxidation, alkylation, deamination, methylation, hydrolysis, nicking, intrachain crosslinks, interchain crosslinks, blunt end strand breaks, staggered end double strand breaks, phosphorylation , dephosphorylation, ubiquitination, glycosylation, single-strand gap, damage by heat, damage by desiccation, damage by UV exposure, damage by gamma radiation, damage by X-radiation , Damage caused by ionizing radiation, Damage caused by non-ionizing radiation, Damage caused by heavy particle radiation, Damage caused by nuclear decay, Damage caused by beta radiation, Damage caused by alpha radiation, Damage caused by neutron radiation damage by proton radiation, damage by cosmic radiation, damage by high pH, damage by low pH, damage by reactive oxidizing species, damage by free radicals, damage by peroxides damage caused by hypochlorite, damage caused by tissue fixation such as formalin or formaldehyde, damage caused by active iron, damage caused by low ionic conditions, damage caused by high ionic conditions damage, damage by unbuffered conditions, damage by nucleases, damage by environmental exposure, damage by fire, damage by mechanical stress, damage by enzymatic degradation, damage by microorganisms, Damage caused by preparative mechanical shearing, damage caused by preparative cleavage, damage that occurs naturally in vivo, damage that occurs during nucleic acid extraction, damage that occurs during sequencing library preparation, damage introduced by polymerases, Damage introduced during nucleic acid repair, damage during nucleic acid end tailing, damage during nucleic acid ligation, damage during sequencing, damage due to mechanical handling of DNA, damage during passage through nanopores damage, damage that occurs as part of aging in living organisms, damage due to chemical exposure of an individual, damage due to mutagens, damage due to carcinogens, damage due to cleavage agents, At least one of damage due to inflammatory damage in vivo caused by oxygen exposure, damage due to breakage of one or more strands, and any combination thereof.

II. Selected Examples of Double Strand Sequencing Methods and Related Adapters and Reagents

Double-stranded sequencing is a method for generating error-corrected DNA sequences from double-stranded nucleic acid molecules, and was originally described in International Patent Publication No. WO 2013/142389 and in US Patent No. 9,752,188 and WO 2017/100441, in Schmitt et.al., PNAS, 2012[1]; in Kennedy et.al., PLOS Genetics, 2013[2]; in Kennedy et.al., Nature Protocols, 2014[3]; and in Schmitt et.al. , described in Nature Methods, 2015 [4]. Each of the aforementioned patents, patent applications, and publications is incorporated herein by reference in its entirety. As shown in Figures 1A-1C, and in certain aspects of the technology, double-stranded sequencing can be used to independently sequence both strands of a single DNA molecule in such a way that in massively parallel sequencing (MPS) During (also commonly referred to as next-generation sequencing (NGS)), derived sequence reads can be identified as originating from the same double-stranded nucleic acid parent molecule, but also differentiated from each other as distinguishable entities after sequencing. The sequence reads obtained from each strand are then compared to obtain an error-corrected sequence of the original double-stranded nucleic acid molecule known as the double-stranded consensus sequence (DCS). The process of double-stranded sequencing makes it possible to unambiguously confirm that both strands of the original double-stranded nucleic acid molecule are represented in the generated sequencing data used to form the DCS.

In certain embodiments, a method of incorporating DS can comprise ligating one or more sequencing adaptors to a target double-stranded nucleic acid molecule comprising a first double-stranded target nucleic acid complex to generate a double-stranded target nucleic acid complex Strand target nucleic acid sequence and second strand target nucleic acid sequence (eg, Figure 22A).

In various embodiments, the resulting target nucleic acid complex can comprise at least one SMI sequence, which may require a degenerate or semi-degenerate sequence applied exogenously (eg, the random double-stranded tag shown in FIG. Sequences identified in Figure 22A as alpha and beta), endogenous information associated with specific cleavage sites for the target double-stranded nucleic acid molecule, or a combination thereof. SMI can make target nucleic acid molecules substantially distinguishable from multiple other molecules in a population that are sequenced alone or in combination with the distinguishing elements of the nucleic acid fragments to which they are attached. Substantially distinguishable features of an SMI element can be carried independently by each single strand forming a double-stranded nucleic acid molecule, such that the derived amplification product of each strand can be identified after sequencing as being derived from the same original substantially unique double-stranded nucleic acid molecule. Stranded nucleic acid molecules. In other embodiments, the SMI may contain additional information and/or may be used for other methods useful for such molecular discrimination functions, such as those described in the above-referenced publications. In another embodiment, the SMI element can be incorporated after adaptor ligation. In some embodiments, the SMI is double-stranded in nature. In other embodiments, it is single-stranded in nature (eg, the SMI can be on the single-stranded portion of the adaptor). In other embodiments, it is a combination of single stranded and double stranded in nature.

In some embodiments, each double-stranded target nucleic acid sequence complex can further comprise an element (eg, an SDE) that allows the amplification products of the two single-stranded nucleic acids that form the target double-stranded nucleic acid molecule to be substantially accessible after sequencing distinguished from each other. In one embodiment, the SDE can include asymmetric primer sites that are included within the sequencing adaptor, or, in other arrangements, sequence asymmetry can be introduced into the adaptor molecule that is not within the primer sequence, such that during amplification And after sequencing, at least one position in the nucleotide sequence of the first strand target nucleic acid sequence complex and the second strand of the target nucleic acid sequence complex differ from each other. In other embodiments, the SMI may include another biochemical asymmetry between the two strands that differs from the standard nucleotide sequence A, T, C, G, or U, but differs in both amplified and sequenced is translated into at least one standard nucleotide sequence difference in the molecule. In yet another embodiment, SDE can be a means of physically separating the two strands prior to amplification, such that the derived amplification products from the first-strand target nucleic acid sequence and the second-strand target nucleic acid sequence remain substantially physically separated from each other, using for the purpose of maintaining the distinction between the two. Other such arrangements or methods for providing SDE functionality that allows differentiation of the first and second strands, such as those described in the above-referenced publications, or other methods that serve the functional purposes described, may be used.

After generating a double-stranded target nucleic acid complex comprising at least one SMI and at least one SDE, or where one or both of these elements are to be subsequently introduced, the complex can undergo DNA amplification, for example with PCR or DNA Any other biochemical method of amplification (eg, rolling circle amplification, multiple displacement amplification, isothermal amplification, bridging amplification, or surface-bound amplification) such that one or more copies of the first-strand target nucleic acid sequence and a or multiple copies of the second strand target nucleic acid sequence (eg, Figure 22B). The one or more amplified copies of the first strand target nucleic acid molecule and the one or more amplified copies of the second target nucleic acid molecule can then undergo DNA sequencing, preferably using a "next-generation" massively parallel DNA sequencing platform (eg, Figure 22B).

Sequence reads generated from first-strand target nucleic acid molecules and second-strand target nucleic acid molecules derived from the original double-stranded target nucleic acid molecule can be identified based on the substantially unique SMIs that are shared and correlated with the opposite-strand target nucleic acid by SDE Molecular differences. In some embodiments, the SMI may be a sequence based on a mathematically error-correcting code (eg, Hamming code), whereby in order to correlate the sequence of the SMI sequence to the complement of the original duplex (eg, double-stranded nucleic acid molecule) For on-strand purposes, some amplification errors, sequencing errors, or SMI synthesis errors are tolerable. For example, for a double-stranded exogenous SMI, where the SMI includes 15 base pairs of the fully degenerate standard DNA base sequence, it is estimated that 4L^15=1,073,741,824 SMI variants will be present in the fully degenerate SMI population. If two SMIs are recovered from reads in a sampled SMI population of 10,000 sequencing data that differ by only one nucleotide in the SMI sequence, the probability of this happening can be calculated mathematically by random chance, and It was decided whether a single base pair difference was more likely to reflect one of the above types of errors, and it was possible to determine that the SMI sequences were in fact derived from the same original double-stranded molecule. In some embodiments wherein the SMI is, at least in part, an exogenously applied sequence, wherein the sequence variants do not fully degenerate from each other, and are at least in part known sequences, in some embodiments the identity of the known sequences may be One or more errors of the foregoing types are designed such that one or more errors of the aforementioned type do not convert the identity of one known SMI sequence to that of another SMI sequence, making it less likely that one SMI will be misinterpreted as another SMI. In some embodiments, the SMI design strategy includes a Hamming code method or a derivative thereof. Once identified, one or more sequence reads generated from the first-strand target nucleic acid molecule are compared to one or more sequence reads generated from the second-strand target nucleic acid molecule to generate an error-corrected sequence of the target nucleic acid molecule (eg, , Figure 22C). For example, a nucleotide position where the bases from a first-strand target nucleic acid sequence and a second-strand target nucleic acid sequence are identical is considered a true sequence, while a nucleotide position that is inconsistent between the two strands is considered a technical error potential sites that can be ignored, eliminated, corrected, or otherwise identified. An error-corrected sequence of the original double-stranded target nucleic acid molecule can thus be generated (shown in Figure 22C). In some embodiments, and after grouping each sequencing read generated from the first-strand target nucleic acid molecule and the second-strand target nucleic acid molecule, respectively, a single-strand can be generated for each of the first-strand and second-strand target nucleic acid molecules chain consensus sequence. Single-stranded consensus sequences from the first-strand target nucleic acid molecule and the second-strand target nucleic acid molecule can then be compared to generate an error-corrected sequence of the target nucleic acid molecule (eg, Figure 22C).

Alternatively, in some embodiments, sites of sequence discordance between the two strands can be identified as potential sites for biologically derived mismatches in the original double-stranded target nucleic acid molecule. Alternatively, in some embodiments, sites of sequence discordance between the two strands can be identified as potential sites for mismatches from DNA synthesis in the original double-stranded target nucleic acid molecule. Alternatively, in some embodiments, sites of sequence inconsistency between the two strands can be identified as potential sites where damaged or modified nucleotide bases are present on one or both strands and are converted into mismatches by an enzymatic process (eg, DNA polymerase, DNA glycosylase, or another nucleic acid-modifying enzyme or chemical process). In some embodiments, this later discovery can be used to infer the presence of nucleic acid damage or nucleotide modifications prior to enzymatic processes or chemical treatments.

In some embodiments, and in accordance with various aspects of the present technology, the sequencing reads generated by the double-stranded sequencing steps discussed herein can be further filtered to eliminate molecules from DNA damage (eg, during storage, transport, in tissue or blood) Sequencing reads during or after extraction, damage during or after library preparation, etc.). For example, DNA repair enzymes, such as uracil-DNA glycosylase (UDG), formamide pyrimidine DNA glycosylase (FPG), and 8-oxoguanine DNA glycosylase (OGG1), can be used to eliminate Or correct for DNA damage (eg, in vitro DNA damage or in vivo damage). For example, these DNA repair enzymes are glycosylases that remove damaged bases from DNA. For example, UDG removes uracil caused by deamination of cytosine (caused by spontaneous hydrolysis of cytosine), and FPG removes 8-oxoguanine (eg, common DNA damage caused by reactive oxygen species). FPG also has lyase activity, which can create a 1-base gap at abasic sites. For example, such abasic sites will typically not subsequently be amplified by PCR due to the inability of the polymerase to replicate the template. Thus, the use of such DNA damage repair/elimination enzymes can effectively remove damaged DNA that is not truly mutated but may otherwise not be detected as errors after sequencing and double-stranded sequence analysis. While in rare cases errors due to damaged bases can usually be corrected by double-stranded sequencing, in theory, complementary errors can occur at the same position on both strands, thus reducing the damage that errors increase The possibility of artifacts can be reduced. Furthermore, during library preparation, some DNA fragments to be sequenced may be single-stranded from their source or from processing steps (eg, mechanical DNA shearing). These regions are typically converted to double-stranded DNA during a "end repair" step known in the art, whereby a DNA polymerase and a nucleoside substrate are added to the DNA sample to extend the 5' recessed ends. Mutagenic sites of DNA damage in the single-stranded portion of the replicated DNA (ie, single-stranded 5' overhangs or internal single-stranded nicks or gaps at one or both ends of the DNA duplex) can be caused during the fill-in reaction Errors that can make single-stranded mutations, synthetic errors, or sites of nucleic acid damage into a double-stranded form that may be misinterpreted as a true mutation in the final double-stranded consensus sequence, whereby a true mutation exists in the original double-stranded nucleic acid molecule, when in fact it does not exist. This condition, known as "pseudoduplex", can be reduced or prevented by the use of such damage destruction/repair enzymes. In other embodiments, this condition can be reduced or eliminated by using strategies that disrupt or prevent the formation of single-stranded portions of the original double-stranded molecule (eg, the use of certain enzymes is used to fragment the original double-stranded nucleic acid material, while Not mechanical shear or some other enzyme that might leave a cut or gap). In other embodiments, the use of processes that eliminate single-stranded portions of the original double-stranded nucleic acid (eg, single-strand-specific nucleases such as S1 nuclease or mung bean nuclease) can be used for similar purposes.

In a further embodiment, the sequencing reads generated by the double-stranded sequencing steps discussed herein can be further filtered to eliminate false mutations by trimming the ends of the reads that are most prone to false-duplex artifacts. For example, DNA fragmentation can generate single-stranded portions at the ends of double-stranded molecules. These single-stranded portions can be filled in during end repair (eg, by Klenow or T4 polymerase). In some cases, polymerases cause replication errors in these end-repaired regions, resulting in the creation of "pseudoduplexes." Once sequenced, the human artifacts of these library preparations can erroneously appear as true mutations. As a result of the end repair mechanism, these errors can be eliminated or reduced from post-sequencing analysis by trimming the ends of the sequenced reads to exclude any mutations that may occur in higher risk regions, thereby reducing the number of false mutations. In one embodiment, such trimming of sequencing reads can be done automatically (eg, normal process steps). In another embodiment, the mutation frequency of the fragment end regions can be assessed, and if a threshold level of mutation is observed in the fragment end regions, sequencing read trimming can be performed prior to generating double-stranded consensus reads of the DNA fragment.

As a specific example, in some embodiments, provided herein are methods of generating error-corrected sequence reads of a double-stranded target nucleic acid material, comprising the steps of: ligating the double-stranded target nucleic acid material to at least one adaptor sequence to form an adaptor- A complex of target nucleic acid material, wherein the at least one adaptor sequence comprises (a) a degenerate or semidegenerate single molecule identifier (SMI) sequence that uniquely labels each molecule of the double-stranded target nucleic acid material, and (b) ) a first nucleotide adaptor sequence of the first strand of the labeled adaptor-target nucleic acid material complex, and a second nucleotide adaptor sequence that is at least partially associated with the labeled adaptor - The first nucleotide sequence of the second strand of the complex of target nucleic acid material is not complementary such that each strand of the complex of adapter-target nucleic acid material has a distinctly recognizable nucleotide sequence relative to its complementary strand. The method can next include amplifying each strand of the adaptor-target nucleic acid material complex to generate a plurality of first-strand adaptor-target nucleic acid complex amplicons and a plurality of second-strand adaptor-target nucleic acid complexes Steps for amplicon. The method may further comprise the step of amplifying the first strand and the second strand to provide the first nucleic acid product and the second nucleic acid product. The method may further comprise the steps of sequencing each of the first nucleic acid product and the second nucleic acid product to generate a plurality of first-strand sequence reads and a plurality of second-strand sequence reads, and identifying at least one first The presence of strand sequence reads and at least one second strand sequence read. The method may further comprise comparing at least one first-strand sequence read to at least one second-strand sequence read, and by ignoring discordant nucleotide positions, or alternatively removing comparisons with one or more nucleotide positions The first and second strand sequence reads are used to generate error-corrected sequence reads of the double-stranded target nucleic acid material, wherein the compared first and second strand sequence reads are non-complementary.

As a further specific example, in some embodiments, provided herein are methods of identifying DNA variants from a sample, comprising the steps of: ligating two strands of nucleic acid material (eg, a double-stranded target DNA molecule) to at least one asymmetric on the adaptor molecule to form an adaptor-target nucleic acid material complex having a first nucleotide sequence and a second nucleotide sequence associated with the first strand (eg, top strand) of the double-stranded target DNA molecule a sequence that is at least partially non-complementary to a first nucleotide sequence associated with the second strand (eg, bottom strand) of the double-stranded target DNA molecule; and each of the amplifying adapter-target nucleic acid material strands, resulting in a distinct but related set of amplified adaptor-target nucleic acid products in each strand. The method may further comprise the step of sequencing each of the plurality of first-strand adaptor-target nucleic acid products and the plurality of second-strand adaptor-target nucleic acid products, confirming that they are from the adaptor-target nucleic acid material complex The presence of at least one amplified sequence read for each strand of the A consensus sequence read of a nucleic acid material (eg, a double-stranded target DNA molecule) of nucleotide bases at which the sequences of the two strands of the nucleic acid material (eg, a double-stranded target DNA molecule) are identical , so that variants occurring at specific positions in the consensus sequence reads (eg, as compared to a reference sequence) are identified as true DNA variants.

In some embodiments, provided herein are methods of generating high-accuracy consensus sequences from double-stranded nucleic acid material, comprising the step of labeling a single double-stranded DNA molecule with an adaptor molecule to form a labeled DNA material, wherein each adaptor molecule comprises (a) a degenerate or semidegenerate single molecule identifier (SMI) that uniquely labels the double-stranded DNA molecule, and (b) first and second non-complementary nucleotide adaptor sequences for each labeled DNA molecule , distinguish the original top strand from the original bottom strand of each individual DNA molecule within the labeled DNA material, and generate a set of replicas of the original top strand of the labeled DNA molecule and the original bottom strand of the labeled DNA molecule A set of replicas to form amplified DNA material. The method may further comprise the steps of generating a first single-stranded consensus sequence (SSCS) from a replica of the original top strand and generating a second single-stranded consensus sequence (SSCS) from a replica of the original bottom strand, One SSCS is compared to the second SSCS of the original bottom strand and generates a high-accuracy consensus sequence with only the nucleotide base at which the sequence of the first SSCS of the original top strand and the original bottom strand the sequence complementarity of the second SSCS.

In further embodiments, provided herein are methods of detecting and/or quantifying DNA damage from a sample comprising double-stranded target DNA molecules comprising ligating the two strands of each double-stranded target DNA molecule to at least one asymmetric adaptor The step of sub-molecules to form a plurality of adaptor-target DNA complexes, wherein each adaptor-target DNA complex has a first nucleotide sequence associated with the first strand of the double-stranded target DNA molecule and a first nucleotide sequence associated with the double-stranded target DNA molecule the second nucleotide sequence associated with the second strand of the target DNA molecule is at least partially non-complementary to the second nucleotide sequence, and for each adaptor-target DNA complex: amplifies the adaptor-target DNA complex of each strand, resulting in each strand generating a distinct but related set of amplified adaptor-target DNA amplicons. The method may further comprise the step of sequencing each of the plurality of first-strand adaptor-target DNA amplicons and the plurality of second-strand adaptor-target DNA amplicons, confirming the origin of the adaptor-target DNA The presence of at least one sequence read in each strand of the complex, and the comparison of the at least one sequence read obtained from the first strand with the at least one sequence read obtained from the second strand to detect and/or quantify nucleotides A base at which a sequence read of one strand of a double-stranded DNA molecule is not identical (eg, not complementary) to a sequence read of the other strand of the double-stranded DNA molecule, allowing detection and/or quantification site of DNA damage. In some embodiments, the method may further comprise the steps of generating a first single-stranded consensus sequence (SSCS) from a first-strand adaptor-target DNA amplicon and generating from a second-strand adaptor-target DNA amplicon Second Single Strand Consensus Sequence (SSCS), compares the first SSCS of the original first strand with the second SSCS of the original second strand, and identifies nucleotides where the sequence of the first SSCS and the sequence of the second SSCS are not complementary bases to detect and/or quantify DNA damage associated with double-stranded target DNA molecules in a sample.

Single Molecule Identifier Sequence (SMI)

According to various embodiments, provided methods and compositions comprise one or more SMI sequences on each strand of nucleic acid material. The SMI can be carried independently by each single strand produced by a double-stranded nucleic acid molecule, such that the derived amplification products of each strand can be identified as derived from the same original substantially unique double-stranded nucleic acid molecule after sequencing. In some embodiments, as those skilled in the art will recognize, SMIs may contain additional information and/or may be used in other methods where such molecular differentiation functions are useful. In some embodiments, the SMI element can be introduced prior to, substantially simultaneously with, or after ligation of the adaptor sequence ligated to the nucleic acid material.

In some embodiments, the SMI sequence can comprise at least one degenerate or semi-degenerate nucleic acid. In other embodiments, the SMI sequence may be non-degenerate. In some embodiments, an SMI can be a sequence associated with or near a fragment end of a nucleic acid molecule (eg, a randomly or semi-randomly cleaved end of a ligated nucleic acid material). In some embodiments, exogenous sequences can be considered in combination with sequences corresponding to the ends of randomly or semi-randomly sheared ligated nucleic acid material (eg, DNA) to obtain SMIs that can distinguish, eg, single DNA molecules from each other sequence. In some embodiments, the SMI sequence is part of an adaptor sequence that is attached to the double-stranded nucleic acid molecule. In certain embodiments, the adaptor sequence that includes the SMI sequence is double-stranded, such that each strand of the double-stranded nucleic acid molecule, after ligation to the adaptor sequence, contains an SMI. In another embodiment, the SMI sequence is single-stranded before or after ligation to a double-stranded nucleic acid molecule, and a complementary SMI sequence can be generated by extending the opposite strand with a DNA polymerase to generate a complementary double-stranded SMI sequence. In other embodiments, the SMI sequence is located in the single-stranded portion of the adaptor (eg, the arms of the adaptor having a Y shape). In such embodiments, the SMI can facilitate the grouping of families of sequence reads derived from the original strand of the double-stranded nucleic acid molecule, and in some cases can confer a relationship between the original first and second strands of the double-stranded nucleic acid molecule (For example, all or some of the SMIs can be associated through a lookup table). In an embodiment, where the first and second strands are labeled with different SMIs, this can be achieved by using one or more endogenous SMIs (eg, fragment-specific features, such as associated with fragment ends of the nucleic acid molecule) or sequences in its vicinity), or use additional molecular tags common to the two original strands (eg, barcodes in the double-stranded portion of the adaptor), or a combination thereof to correlate sequence reads from the two original strands. In some embodiments, each SMI sequence can comprise between about 1 and about 30 nucleic acids (eg, 1, 2, 3, 4, 5, 8, 10, 12, 14, 16, 18, 20 or more degenerate or semi-degenerate nucleic acids).

In some embodiments, the SMI can be attached to one or both of the nucleic acid material and the adaptor sequence. In some embodiments, SMIs can be attached to T-overhangs, A-overhangs, CG-overhangs, including those of known nucleotide lengths (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) "sticky ends" or overhangs of single-stranded overhang regions on at least one of the end, the dehydroxylated base, and the blunt end.

In some embodiments, SMI sequences can be considered (designed) in conjunction with (or based on) sequences corresponding to, for example, randomly or semi-randomly spliced ends of nucleic acid material (eg, linked nucleic acid material) to obtain a single nucleic acid molecule capable of combining SMI sequences that are distinguished from each other.

In some embodiments, the at least one SMI can be an endogenous SMI (eg, an SMI associated with a cleavage point (eg, a fragment end), eg, defined using the cleavage point itself or using nucleic acid material immediately adjacent to the cleavage point number of nucleotides [eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides from the cleavage point]). In some embodiments, the at least one SMI can be an exogenous SMI (eg, an SMI that includes sequences not found on the target nucleic acid material).

In some embodiments, the SMI may be or include an imaging moiety (eg, a fluorescent or otherwise optically detectable moiety). In some embodiments, such SMI allows detection and/or quantification without the need for an amplification step.

In some embodiments, an SMI element can include two or more different SMI elements located at different positions on the adaptor-target nucleic acid complex.

Various embodiments of SMI are further disclosed in International Patent Publication No. WO2017/100441, the entire contents of which are incorporated herein by reference.

Chain Definition Element (SDE)

In some embodiments, each strand of the double-stranded nucleic acid material may further comprise an element that renders the amplification products of the two single-stranded nucleic acids forming the target double-stranded nucleic acid material substantially distinguishable from each other after sequencing. In some embodiments, the SDE may be or include an asymmetric primer site within the sequencing adaptor, or, in other arrangements, sequence asymmetry may be introduced into the adaptor sequence rather than the primer sequence, such that in After amplification and sequencing, at least one position in the nucleotide sequence of the first strand target nucleic acid sequence complex and the second strand of the target nucleic acid sequence complex differ from each other. In other embodiments, the SDE may include another biochemical asymmetry between the two strands that differs from the standard nucleotide sequence A, T, C, G, or U, but differs in both amplified and sequenced Molecules are converted to at least one standard nucleotide sequence difference. In yet another embodiment, the SDE may be or include a means of physically separating the two strands prior to amplification such that the derived amplification products from the first-strand target nucleic acid sequence and the second-strand target nucleic acid sequence remain substantially physically separated from each other, For the purpose of maintaining the distinction between the two derived amplification products. Other such arrangements or methods for providing SDE functionality that allow differentiation of the first and second strands can be utilized.

In some embodiments, the SDE may be capable of forming loops (eg, hairpin loops). In some embodiments, the loop can include at least one endonuclease recognition site. In some embodiments, the target nucleic acid complex may contain an endonuclease recognition site that facilitates intra-loop cleavage events. In some embodiments, loops may include non-standard nucleotide sequences. In some embodiments, the included non-standard nucleotides can be recognized by one or more enzymes that facilitate strand cleavage. In some embodiments, the included non-standard nucleotides can be targeted by one or more chemical processes that facilitate cleavage of strands in the loop. In some embodiments, the loops can contain modified nucleic acid linkers that can be targeted by one or more enzymatic, chemical, or physical processes that facilitate cleavage of strands in the loop. In some embodiments, such modified linkers are photocleavable linkers.

Various other molecular tools are available as SMI and SDE. In addition to cleavage sites and DNA-based labels, single-molecule compartmentalization approaches that maintain physical proximity of paired strands or other non-nucleic acid labeling approaches can perform strand-associated functions. Similarly, asymmetric chemical labeling of the adaptor strands in such a way that the adaptor strands can be physically separated can function as an SDE. A recently described variant of double-stranded sequencing uses bisulfite transformation to convert naturally occurring strand asymmetry in the form of cytosine methylation into sequence differences that distinguish the two strands. Although this embodiment limits the types of mutations that can be detected, exploiting the concept of natural asymmetry is noteworthy in the context of emerging sequencing technologies that can directly detect modified nucleotides. Various embodiments of SDE are further disclosed in International Patent Publication No. WO2017100441, the entire contents of which are incorporated by reference.

Adapters and Adapter Sequences

In various arrangements, adaptor molecules including SMIs (eg, molecular barcodes), SDEs, primer sites, flow cell sequences, and/or other features are contemplated for use in many of the embodiments disclosed herein. In some embodiments, the provided adaptors can be or include one or more sequences that are complementary or at least partially complementary to PCR primers (eg, primer sites) having at least one of the following properties: 1 ) high target specificity; 2) capable of being multiplexed; and 3) amplification that exhibits robustness and minimal bias.

In some embodiments, adaptor molecules can be "Y" shaped, "U" shaped, "hairpin" shaped, have bubbles (eg, non-complementary portions of the sequence) or other features. In other embodiments, the adaptor molecule may comprise a "Y" shape, a "U" shape, a "hairpin" shape, or a bubble. Certain adaptors may include modified or non-standard nucleotides, restriction sites, or other features for manipulation of structure or function in vitro. Adaptor molecules can be ligated to a variety of nucleic acid materials with termini. For example, adaptor molecules may be suitable for ligation to T-overhangs, A-overhangs, CG-overhangs, polynucleotide overhangs (also referred to herein as "sticky ends" or "sticky overhangs"), Dehydroxylated bases, blunt ends of nucleic acid material, and ends of molecules where the 5' of the target is dephosphorylated or otherwise blocked from conventional ligation. In other embodiments, the adaptor molecule may contain modifications on the 5' strand of the ligation site that dephosphorylate or otherwise prevent ligation. In the latter two examples, such a strategy can be used to prevent dimerization of library fragments or adaptor molecules.

In some embodiments, the adaptor molecule can include a capture moiety suitable for isolating the desired target nucleic acid molecule to which it is attached.

Adapter sequences may refer to single-stranded sequences, double-stranded sequences, complementary sequences, non-complementary sequences, partially complementary sequences, asymmetric sequences, primer binding sequences, flow cell sequences, linker sequences, or other sequences provided by the adaptor molecule. In certain embodiments, an adaptor sequence may refer to a sequence used for amplification by means of complementary oligonucleotides.

In some embodiments, provided methods and compositions comprise at least one adaptor sequence (eg, two adaptor sequences, one on each of the 5' and 3' ends of the nucleic acid material). In some embodiments, provided methods and compositions can include 2 or more adaptor sequences (eg, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, at least two of the adaptor sequences differ from each other (eg, by sequence). In some embodiments, each adaptor sequence differs from each other (eg, by sequence). In some embodiments, at least one adaptor sequence is at least partially non-complementary to at least a portion of at least one other adaptor sequence (eg, non-complementary to at least one nucleotide).

In some embodiments, the adaptor sequence includes at least one non-standard nucleotide. In some embodiments, the non-standard nucleotide is selected from the group consisting of abasic sites, uracil, tetrahydrofuran, 8-oxo-7,8-dihydro-2'deoxyadenosine (8-oxo-A), 8-oxo-7,8-dihydro2'-deoxyguanosine (8-oxo-G), deoxyinosine, 5'nitroindole, 5-hydroxymethyl-2'-deoxycytidine, Isocytosine, 5'-methylisocytosine or isoguanosine, methylated nucleotides, RNA nucleotides, ribonucleotides, 8-oxoguanine, photocleavable linkers, biotinylation nucleotides, desthiobiotin nucleotides, thiol-modified nucleotides, acrylate-modified nucleotides, iso-dC, iso-dG, 2'-O-methyl nucleotides, inosine nucleosides Acid-locked nucleic acid, peptide nucleic acid, 5-methyl dC, 5-bromodeoxyuridine, 2,6-diaminopurine, 2-aminopurine nucleotide, abasic nucleotide, 5-nitroindole nucleoside acid, adenylated nucleotides, azide nucleotides, digoxigenin nucleotides, I-linkers, 5'hexynyl modified nucleotides, 5-octadiynyl dU, photocleavable spacers, non-photocleavable spacers, click chemistry compatible modified nucleotides, and any combination thereof.

In some embodiments, the adaptor sequence includes a moiety having magnetic properties (ie, a magnetic moiety). In some embodiments, this magnetic property is paramagnetic. In some embodiments, wherein the adaptor sequence includes a magnetic moiety (eg, a nucleic acid material attached to an adaptor sequence that includes a magnetic moiety), when a magnetic field is applied, the adaptor sequence that includes the magnetic moiety is substantially different from the adaptor sequence that does not include the magnetic moiety ( For example, adaptor sequences linked to nucleic acid material that do not contain adaptor sequences that do not have magnetic moieties) are isolated.

In some embodiments, at least one adaptor sequence is located 5' to the SMI. In some embodiments, at least one adaptor sequence is located 3' to the SMI.

In some embodiments, the adaptor sequence can be linked to at least one of the SMI and the nucleic acid material through one or more linker domains. In some embodiments, the linker domain may consist of nucleotides. In some embodiments, the linker domain may comprise at least one modified nucleotide or non-nucleotide molecule (eg, as described elsewhere in this disclosure). In some embodiments, the linker domain can be or include a loop.

In some embodiments, the adaptor sequences on either or both ends of each strand of the double-stranded nucleic acid material may further comprise one or more SDE-providing elements. In some embodiments, the SDE can be or include an asymmetric primer site included in the adaptor sequence.

In some embodiments, an adaptor sequence can be or include at least one SDE and at least one ligation domain (ie, a domain that is modified according to the activity of at least one ligase, eg, suitable for attachment to a nucleic acid by the activity of the ligase). material domains). In some embodiments, from 5' to 3', the adaptor sequence can be or include a primer binding site, an SDE, and a ligation domain.

Various methods for synthesizing double-stranded sequencing adapters have been previously described in, for example, US Patent No. 9,752,188, International Patent Publication No. WO2017/100441, and International Patent Application No. PCT/US18/59908 (filed on November 8, 2018) are described in , the entire contents of all of these patents are incorporated herein by reference.

primer

In some embodiments, one or more PCR primers having at least one of the following properties are contemplated for use in various embodiments in accordance with various aspects of the present technology: 1) high target specificity; 2) capable of being used by multiplex; and 3) amplification that exhibits robustness and minimal bias. A number of previous research and commercial products have been designed to meet primer mixes for some of these criteria for conventional PCR-CE. However, it has been noted that these primer mixes are not always the best choice for use with MPS. In fact, developing highly multiplexed primer mixes can be a challenging and time-consuming process. Conveniently, both Illumina and Promega have recently developed multiplex-compatible primer mixes for the Illumina platform that have shown robust and efficient amplification of a variety of standard and non-standard STR and SNP loci. Because these kits use PCR to amplify their target regions prior to sequencing, the 5' end of each read in the paired-end sequencing data corresponds to the 5' end of the PCR primers used to amplify the DNA. In some embodiments, provided methods and compositions comprise primers designed to ensure uniform amplification, which may require changes in reaction concentrations, melting temperatures, and minimize secondary structure and intra-/inter-primer interactions change. Various techniques have been described for highly multiplex primer optimization for MPS applications. In particular, these techniques are commonly referred to as ampliseq methods, as described in the art.

Amplify

In various embodiments, the provided methods and compositions utilize or are used for at least one amplification step, wherein nucleic acid material (or a portion thereof, eg, a specific target region or locus) is amplified to form amplified nucleic acid material (eg, some amplicon products).

In some embodiments, amplifying the nucleic acid material comprises the step of amplifying nucleic acid material derived from each of the first and second nucleic acid strands from the original double-stranded nucleic acid material using at least one single-stranded oligonucleotide, whereby The single-stranded oligonucleotide is at least partially complementary to the sequence present in the first adaptor sequence such that the SMI sequence is at least partially retained. The step of amplifying further comprises amplifying each relevant strand using a second single-stranded oligonucleotide, and such second single-stranded oligonucleotide may (a) be at least partially complementary to the relevant target sequence, or ( b) is at least partially complementary to a sequence present in the second adaptor sequence such that the at least one single-stranded oligonucleotide and the second single-stranded oligonucleotide are oriented in a manner effective to amplify the nucleic acid material.

In some embodiments, the nucleic acid material in the amplified sample may comprise nucleic acid material in amplification "tubes" (eg, PCR tubes), emulsion droplets, microchambers, and other examples above or in other known containers. In some embodiments, the amplified nucleic acid material can be included in two or more (eg, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more) sample) to amplify nucleic acid material in physically separated samples (eg, tubes, droplets, chambers, containers, etc.). For example, the initial sample can be divided into multiple containers prior to the amplification step. In some embodiments, each sample contains substantially the same amount of amplified nucleic acid material as each other sample, and in some embodiments, at least two samples contain substantially different amounts of amplified nucleic acid material.

In some embodiments, at least one amplification step includes at least one primer that is or includes at least one non-standard nucleotide. In some embodiments, the non-standard nucleotides are selected from the group consisting of uracil, methylated nucleotides, RNA nucleotides, ribonucleotides, 8-oxoguanine, biotinylated nucleotides, locked nucleic acids, Peptide nucleic acids, high Tm nucleic acid variants, allelic recognition nucleic acid variants, any other nucleotide or linker variants described elsewhere herein, and any combination thereof.

While any amplification reaction suitable for use is considered compatible with some embodiments, by way of specific example, in some embodiments, the amplification step may be or include polymerase chain reaction (PCR), rolling circle amplification ( RCA), multiple displacement amplification (MDA), isothermal amplification, polymerase clonal amplification in emulsion, bridging amplification on a surface, on the surface of a bead, or within a hydrogel, and any combination thereof.

In some embodiments, amplifying the nucleic acid material comprises using single-stranded oligonucleotides that are at least partially bound to adaptor sequences on the 5' and 3' ends of each strand of the nucleic acid material Regional complementarity. In some embodiments, amplifying the nucleic acid material comprises using at least one single-stranded oligo that is at least partially complementary to the target region or an associated target sequence (eg, genomic sequence, mitochondrial sequence, plasmid sequence, synthetically produced target nucleic acid, etc.). Nucleotides and single-stranded oligonucleotides that are at least partially complementary to regions of the adaptor sequence (eg, primer sites).

的PCR优化试剂盒，其含有许多预先配制的缓冲液，这些缓冲液被部分优化用于各种PCR应用，例如多重、实时、富含GC和抑制剂抗性扩增。这些预先配制的缓冲液可以快速地补充有不同的Mg²⁺和引物浓度，以及引物池比率。此外，在一些实施例中，可以评估和/或使用各种循环条件(例如，热循环)。在评估特定的实施例是否适合特定的期望应用时，可以评估特异性、杂合基因座的等位基因覆盖率、基因座间平衡和深度以及其他方面中的一个或多个。扩增成功的测量可以包含产物的DNA测序、通过凝胶或毛细管电泳或HPLC或其他大小分离方法对产物的评价，随后是片段可视化、使用双链核酸结合染料或荧光探针的熔融曲线分析、质谱或本领域已知的其他方法。In general, robust amplification, such as PCR amplification, can be highly dependent on reaction conditions. For example, multiplex PCR pair buffer composition, monovalent or divalent cation concentration, detergent concentration, crowding agent (i.e. PEG, glycerol, etc.) concentration, primer concentration, primer Tms, primer design, primer GC content, primer modified nucleotides Properties and cycling conditions (ie, temperature and extension time and rate of temperature change) can be sensitive. Optimization of buffer conditions can be a difficult and time-consuming process. In some embodiments, the amplification reaction can use at least one of buffers, primer pool concentrations, and PCR conditions according to previously known amplification protocols. In some embodiments, new amplification protocols can be created, and/or amplification reaction optimizations can be used. As a specific example, in some embodiments, PCR optimization kits can be used, such as from

The PCR Optimization Kits from ® contain a number of pre-formulated buffers that are partially optimized for various PCR applications such as multiplex, real-time, GC-rich, and inhibitor-resistant amplification. These pre-formulated buffers can be quickly supplemented with different Mg ²⁺ and primer concentrations, as well as primer pool ratios. Additionally, in some embodiments, various cycling conditions (eg, thermal cycling) may be evaluated and/or used. In assessing the suitability of a particular embodiment for a particular desired application, one or more of specificity, allelic coverage of heterozygous loci, balance and depth between loci, and other aspects can be assessed. Measurement of amplification success can include DNA sequencing of the product, evaluation of the product by gel or capillary electrophoresis or HPLC or other size separation methods, followed by fragment visualization, melting curve analysis using double-stranded nucleic acid binding dyes or fluorescent probes, Mass spectrometry or other methods known in the art.

According to various embodiments, any of a variety of factors can affect the length of a particular amplification step (eg, the number of cycles in a PCR reaction, etc.). For example, in some embodiments, the provided nucleic acid material may be damaged or otherwise suboptimal (eg, degraded and/or contaminated). In such cases, longer amplification steps may help to ensure that the desired product is amplified to an acceptable degree. In some embodiments, the amplification step can provide an average of 3 to 10 sequenced PCR copies from each starting DNA molecule, although in other embodiments only a single copy of each of the first and second strands is required copy. Without wishing to be bound by a particular theory, too many or too few PCR copies may result in reduced assay efficiency and ultimately reduced depth. Typically, the number of nucleic acid (eg, DNA) fragments used in an amplification (eg, PCR) reaction is a major adjustable variable that can determine the number of reads that share the same SMI/barcode sequence.

Nucleic acid material

type

According to various embodiments, any of a variety of nucleic acid materials may be used. In some embodiments, the nucleic acid material can include at least one modification to a polynucleotide within a typical sugar-phosphate backbone. In some embodiments, the nucleic acid material can include at least one modification within any base in the nucleic acid material. For example, by way of non-limiting example, in some embodiments, the nucleic acid material is or includes at least one of double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA) A sort of.

source

It is contemplated that the nucleic acid material can be derived from any of a variety of sources. For example, in some embodiments, nucleic acid material is provided from a sample from at least one subject (eg, a human or animal subject) or other biological source. In some embodiments, the nucleic acid material is provided from an inventory/stored sample. In some embodiments, the sample is or includes blood, serum, sweat, saliva, cerebrospinal fluid, mucus, uterine lavage, vaginal swabs, nasal swabs, buccal swabs, tissue scrapings, hair, fingerprints, urine , feces, vitreous humor, peritoneal lavage, sputum, bronchial lavage, oral lavage, pleural lavage, gastric lavage, gastric juice, bile, pancreatic lavage, bile duct lavage, common bile duct Irrigation fluid, gallbladder fluid, synovial fluid, infected wounds, uninfected wounds, archaeological samples, forensic samples, water samples, tissue samples, food samples, bioreactor samples, plant samples, nail scrapings, semen, prostate fluid , tubal lavage fluid, cell-free nucleic acid, intracellular nucleic acid, metagenomic samples, lavage fluid for implanted foreign bodies, nasal lavage fluid, intestinal fluid, epithelial brushing, epithelial lavage fluid, tissue biopsy samples, autopsy At least one of samples, necropsy samples, organ samples, human identification samples, artificially generated nucleic acid samples, synthetic genetic samples, nucleic acid data storage samples, tumor tissue, and any combination thereof. In other embodiments, the sample is or includes at least one of a microorganism, a plant-based organism, or any collected environmental sample (eg, water, soil, archaeology, etc.).

retouch

According to various embodiments, the nucleic acid material may undergo one or more modifications before, substantially simultaneously, or after any particular step, depending on the application for which the particular provided method or composition is used.

In some embodiments, the modification may be or include repair of at least a portion of the nucleic acid material. Although any means of nucleic acid repair suitable for use are considered compatible with some embodiments, certain exemplary methods and compositions are thus described below and in the examples.

By way of non-limiting example, in some embodiments, DNA repair enzymes such as uracil-DNA glycosylase (UDG), formamide pyrimidine DNA glycosylase (FPG), and 8-oxoguanine can be utilized DNA glycosylase (OGG1), to correct DNA damage (eg, in vitro DNA damage). As discussed above, these DNA repair enzymes are, for example, glycosylases that remove damaged bases from DNA. For example, UDG removes uracil caused by deamination of cytosine (caused by spontaneous hydrolysis of cytosine), and FPG removes 8-oxoguanine (eg, the most common DNA damage caused by reactive oxygen species). FPG also has lyase activity, which can create a 1-base gap at abasic sites. Such abasic sites would then not be able to be amplified by PCR, for example, because the polymerase cannot replicate the template. Thus, the use of such DNA damage repair enzymes can effectively remove damaged DNA without true mutations, but may not otherwise be detected as errors after sequencing and duplex sequence analysis.

As discussed above, in further embodiments, the sequencing reads generated from the processing steps described herein can be further filtered to eliminate false mutations by trimming the ends of the reads that are most prone to artifacts. For example, DNA fragmentation can generate single-stranded portions at the ends of double-stranded molecules. These single stranded moieties can be filled in during end repair (eg, by Klenow). In some cases, polymerases cause replication errors in these end-repaired regions, resulting in the creation of "pseudoduplexes." Once sequenced, these artifacts may appear to be true mutations. As a result of the end-repair mechanism, these errors can be eliminated from post-sequencing analysis by trimming the ends of the sequenced reads to exclude any mutations that may have occurred, thereby reducing the number of erroneous mutations. In some embodiments, such trimming of sequencing reads can be done automatically (eg, normal process steps). In some embodiments, the frequency of mutations in the fragment end regions can be assessed, and if a threshold level of mutation is observed in the fragment end regions, sequencing read trimming can be performed prior to generating double-stranded consensus reads of the DNA fragment.

Some embodiments of the DS method provide a PCR-based targeted enrichment strategy compatible with the use of molecular barcodes for error correction. For example, the Sequencing Enrichment Strategy for Sequencing ("SPLiT-DS") method step utilizing isolated PCR of ligated templates may also benefit from the use of pre-enriched nucleic acid material of one or more of the embodiments described herein. SPLiT-DS was originally described in International Patent Publication No. WO/2018/175997 (the entire contents of which are incorporated herein by reference). The SPLiT-DS method can start from double-stranded nucleic acid material (eg, from a DNA sample) fragmented with molecular barcode labelling (eg, tagging) in a similar manner to that described above and with reference to standard DS library construction protocols . In some embodiments, the double-stranded nucleic acid material can be fragmented (eg, such as with cell-free DNA, damaged DNA, etc.); however, in other embodiments, the various steps can involve the use of mechanical shearing such as acoustic Treatment or other DNA cleavage methods (such as described further herein) to fragment nucleic acid material. Aspects of labeling the fragmented double-stranded nucleic acid material may include end repair and 3'-dA-tailing (if desired in a particular application) followed by ligation of the double-stranded nucleic acid fragments with SMI-containing DS adapters. In other embodiments, the SMI can be an endogenous sequence, or a combination of exogenous and endogenous sequences, used to uniquely correlate information from the two strands of the original nucleic acid molecule. After ligation of the adaptor molecule to the double-stranded nucleic acid material, the method can proceed to amplification (eg, PCR amplification, rolling circle amplification, multiple displacement amplification, isothermal amplification, bridging amplification, surface-bound amplification, etc. ).

In certain embodiments, primers specific for, for example, one or more adaptor sequences can be used to amplify each strand of nucleic acid material, thereby producing a nucleic acid amplicon derived from each strand of the original double-stranded nucleic acid molecule. Multiple copies where each amplicon retains the originally associated SMI. Following the relevant steps of amplification and removal of reaction by-products, the sample can be (preferably, but not necessarily, substantially uniformly) divided into two or more separate samples (eg, in a tube, in an emulsion in droplets, in microchambers, in separate droplets on a surface, or in other known containers, collectively referred to as "tubes"). After isolation, and according to one embodiment of the SPLiT-DS process, the method may comprise amplifying the first strand in the first sample by using primers specific for the first adaptor sequence to provide the first nucleic acid product, and by The second strand in the second sample is amplified using primers specific for the second adaptor sequence to provide a second nucleic acid product. Next, the method can comprise sequencing each of the first nucleic acid product and the second nucleic acid product, and comparing the sequence of the first nucleic acid product to the sequence of the second nucleic acid product. In some embodiments, the nucleic acid material includes adaptor sequences on each of the 5' and 3' ends of each strand of the nucleic acid material. In certain applications, amplification of individual strands in an isolated sample can be accomplished using single-stranded oligonucleotides that are at least partially complementary to associated target sequences, such that single-molecule identifier sequences are at least partially preserved.

Examples of applied selections

As described herein, the provided methods and compositions can be used for any one of a variety of purposes and/or any one of a variety of situations. For the purpose of specific illustration only, non-limiting examples of applications and/or situations are described below.

Monitoring response to therapy (tumor mutations, etc.)

The advent of next-generation sequencing (NGS) in genomic research has enabled the characterization of tumor mutational landscapes in unprecedented detail and has led to diagnostic, prognostic, and classification of clinically actionable mutations. Collectively, these mutations hold great promise for improving cancer outcomes through personalized medicine and for potential early cancer detection and screening. Prior to this disclosure, a key limitation in the field was that these mutations could not be detected when they were present at low frequencies. Clinical biopsies are often composed primarily of normal cells, and detecting cancer cells based on their DNA mutations is a technical challenge even for modern NGS. Identifying tumor mutations in thousands of normal genomes is akin to finding a needle in a haystack, requiring levels of sequencing accuracy that exceed previously known methods.

In general, this problem is exacerbated in the context of liquid biopsies, where the challenge is not only to provide the extreme sensitivity needed to find tumor mutations, but also to do so with the minimal amount of DNA typically present in these biopsies a little. The term 'liquid biopsy' generally refers to the ability of blood to tell cancer based on the presence of circulating tumor DNA (ctDNA). ctDNA is released by cells into the bloodstream and has shown great promise for monitoring, detecting and predicting cancer, as well as for tumor genotyping and therapy selection. These applications may revolutionize the current management of patients with cancer, however, progress has been slower than previously expected. The main problem is that ctDNA typically represents only a small fraction of all cell-free DNA (cfDNA) present in plasma. In metastatic cancers, its frequency may be >5%, but in localized cancers, its frequency is only between 1% and 0.001%. In theory, DNA subpopulations of any size can be detected by assaying a sufficient number of molecules. However, a fundamental limitation of previous methods is the high frequency with which bases are incorrectly scored. Errors typically occur during cluster generation, sequencing cycles, poor cluster resolution, and template degradation. The result is that about 0.1-1% of the sequenced bases are called incorrectly. Further problems can arise from polymerase errors and amplification bias during PCR, which can lead to population skew or introduce false mutant allele frequencies (MAFs). In conclusion, none of the previously known techniques, including conventional NGS, can perform at the level required to detect low-frequency mutations.

Due to its high accuracy, DS and methods for increasing the conversion and workflow efficiency of these sequencing platforms hold promise in the field of oncology. As described herein, the provided methods and compositions allow for innovative approaches to DS methods that integrate double-stranded molecular labeling of DS with target nucleic acid enrichment for increased efficiency and scalability while maintaining error correction .

In addition to the need for highly accurate and efficient assays, the realities of clinical laboratories also demand assays that are fast, scalable, and reasonably cost-effective. Accordingly, various embodiments that improve workflow efficiency of DSs (eg, enrichment strategies for DSs) in accordance with aspects of the present technology are highly desirable. As described herein, digestion/size selection enrichment and affinity-based enrichment of specific target sequences for DS applications provides high target specificity, low DNA input performance, scalability, and minimal cost.

Some embodiments of the provided methods and compositions are particularly important for cancer research in general, and for the ctDNA field, because the technology developed herein has the potential to identify cancer mutations with unprecedented sensitivity, while enabling DNA input, preparation time, and Cost minimization. The target nucleic acid enrichment embodiments disclosed herein can be used in clinical applications that can significantly increase survival through improved patient management and early cancer detection.

patient stratification

Patient stratification, which generally refers to the division of patients based on one or more non-treatment-related factors, is a topic of great interest in the medical community. Much of this concern may be due to the fact that some therapeutic candidates have failed to gain FDA approval, in part due to previously unrecognized differences between patients in trials. These differences may be or include one or more genetic differences that result in a therapeutic agent being metabolized differently, or side effects that occur or are exacerbated in one group of patients relative to one or more groups of other patients. In some cases, some or all of these differences can be detected as one or more different genetic characteristics in a patient that result in a different response to a therapeutic agent than in other patients who do not exhibit the same genetic characteristics.

Thus, in some embodiments, the provided methods and compositions can be used to determine which subject or subjects in a particular patient population (eg, patients with a common disease, disorder, or condition) may respond to a particular therapy. For example, in some embodiments, provided methods and/or compositions can be used to assess whether a particular subject has a genotype associated with poor response to therapy. In some embodiments, provided methods and/or compositions can be used to assess whether a particular subject has a genotype associated with a positive response to therapy.

Forensic Science

Previous approaches to forensic DNA analysis have relied almost exclusively on capillary electrophoretic separation of PCR amplicons to identify length polymorphisms in short tandem repeats. Since its introduction in 1991, this type of analysis has proven extremely valuable. Since then, several publications have introduced standardized protocols, validated their use in laboratories around the world, detailed its use in many different population groups, and introduced more efficient methods such as miniSTR .

While this approach has proven very successful, the technique has a number of drawbacks that limit its usefulness. For example, current methods of STR genotyping often produce background signals caused by PCR shadow bands caused by polymerase slippage on template DNA. This problem is especially important in samples with more than one contributor because it is difficult to distinguish between shadow band alleles and true alleles. Another problem arises when analyzing degraded DNA samples. Variations in fragment length often result in significantly reduced, or even absent, longer PCR fragments. Consequently, profiles from degraded DNA typically have lower discriminative power.

The introduction of MPS systems has the potential to address several challenging problems in forensic analysis. For example, these platforms offer unparalleled capabilities to allow simultaneous analysis of nuclear and mtDNA STRs and SNPs, which will greatly increase the ability to discriminate between individuals and offer the possibility to determine ethnicity and even physical attributes. Furthermore, unlike PCR-CE, which simply reports the average genotype of an aggregated population of molecules, the MPS technique numerically tabulates the complete nucleotide sequence of many individual DNA molecules, providing the ability to detect MAFs in heterogeneous DNA mixtures unique ability. As forensic specimens that include two or more contributors remain one of the most problematic issues in forensic science, the impact of MPS on the field of forensic science can be enormous.

The publication of the human genome highlights the enormous power of the MPS platform. However, until recently, the full capabilities of these platforms had limited application to forensics because read lengths were significantly shorter than STR loci, precluding the ability to call length-based genotypes. Initially, pyrosequencers such as the Roche 454 platform were the only platforms with sufficient read length to sequence core STR loci. However, read lengths in competing technologies have increased, giving their utility in forensic applications a role. Numerous studies have revealed the potential for MPS genotyping of STR loci. Overall, the overall result of all these studies (regardless of platform) is that STRs can be successfully typed, resulting in genotypes comparable to CE analysis, even from compromised forensic samples.

While all of these studies show agreement with traditional PCR-CE methods and even show additional benefits such as detection of SNPs within STRs, they also highlight some of the current problems with this technique. For example, current MPS methods for STR genotyping rely on multiplex PCR to provide enough DNA to sequence and introduce PCR primers. However, because multiplex PCR kits are designed for PCR-CE, they contain primers for amplicons of different sizes. This variation results in an imbalance in coverage, favoring the amplification of smaller fragments, which can lead to loss of alleles. In fact, recent studies have shown that differences in PCR efficiency can affect mixture components, especially at low MAF. To address this issue, several sequencing kits specifically designed for use in forensics are now commercially available, and validation studies are beginning to be reported. However, amplification bias is still evident due to high levels of multiplexing.

Like PCR-CE, MPS cannot avoid PCR shadow bands. The vast majority of MPS studies on STRs report the occurrence of artificially inserted alleles. Recently, a systematic MPS study reported that most shadow band events manifested as shorter length polymorphisms that differed from true alleles in four base pair units, the most common of which was n-4, but which The n-8 and n-12 positions were also observed. The percentage of shadow bands typically occurs in about 1% of reads, but may be as high as 3% at some loci, suggesting that MPS can display shadow bands at a higher rate than PCR-CE.

Rather, in some embodiments, the provided methods and compositions allow for high-quality and efficient sequencing of low-quality and/or small amounts of samples, as described above and in the Examples below. Thus, in some embodiments, provided methods and/or compositions may be used for rare variant detection from DNA of one individual mixed in low abundance with DNA of another individual of a different genotype.

Forensic DNA samples often contain non-human DNA. Potential sources of such exogenous DNA are: the source of the DNA (eg, microorganisms in saliva or buccal samples), the surface environment where the sample was collected, and contamination from the laboratory (eg, reagents, work area, etc.). Another aspect provided by some embodiments is that certain provided methods and compositions allow contaminating nucleic acid material to be distinguished from other sources (eg, different species) and/or surface or environmental contaminants such that these materials (and /or their effects) can be removed from the final analysis without biasing the sequencing results.

In highly degraded DNA, locus-specific PCR may not work well because the DNA fragments do not contain the necessary primer annealing sites, resulting in allelic deletions. This situation will limit the uniqueness of genotype calls and the confidence in matching is less guaranteed, especially in mixed trials. However, in some embodiments, the provided methods and compositions allow for the use of single nucleotide polymorphisms (SNPs) in addition to or in place of STR markers.

In fact, SNPs are increasingly relevant for forensic work as data on human genetic variation continues to increase. As such, in some embodiments, the provided methods and compositions use primer design strategies such that multiplex primer plates can be created, eg, based on currently available sequencing kits, that actually ensure that reads traverse one or more SNP positions.

further example

1. A method for enriching target nucleic acid material, comprising:

provide nucleic acid material;

cleaving the nucleic acid material with one or more targeted endonucleases such that a target region of a predetermined length is separated from the remainder of the nucleic acid material;

Enzymatic destruction of non-targeted nucleic acid material;

release a target region of predetermined length from the targeted endonuclease; and

The cleaved target region is analyzed.

2. The method of example 1, wherein enzymatically destroying the non-targeted nucleic acid material comprises providing an exonuclease.

3. The method of example 1, wherein enzymatically destroying the non-targeted nucleic acid material comprises providing one or more of an exonuclease and an endonuclease.

4. The method of example 1, wherein disrupting comprises at least one of enzymatic digestion and enzymatic cleavage.

5. The method of any one of examples 1-4, wherein the one or more targeted endonucleases remain bound to the target region during the enzymatic destruction step.

6. The method according to any one of examples 1-5, wherein at least one targeted endonuclease is a ribonucleoprotein complex comprising a capture label, and wherein a target region of a predetermined length is linked to the nucleic acid by capturing the label. The remainder are physically separated, while at least one targeted endonuclease remains bound to the target region.

7. The method according to example 1-5, wherein at least one targeted endonuclease is a ribonucleoprotein complex comprising a capture label, and wherein the method further comprises using an extraction portion configured to bind the capture label Capture the target area.

8. The method of example 6 or example 7, wherein the capture label is or comprises at least one of acridine, azide, azide (NHS ester), digoxigenin (NHS ester), I-Linker, Amino Modifier C6, Amino Modifier C12, Amino Modifier C6 dT, Unilink Amino Modifier, Hexynyl, 5-Octadiynyl dU, Biotin, Biotin (azide) ), biotin dT, biotin TEG, dual biotin, PC biotin, desthiobiotin TEG, thiol modifier C3, dithiol, thiol modifier C6 S-S, succinyl group.

9. The method of example 7, wherein the extraction moiety is or comprises aminosilane, epoxysilane, isothiocyanate, aminophenylsilane, aminopropylsilane, mercaptosilane, aldehyde, epoxide, phosphonic acid At least one of esters, streptavidin, avidin, haptens that recognize antibodies, specific nucleic acid sequences, magnetically attractive particles (Dynabeads), and photolabile resins.

10. The method of example 7, wherein the extraction moiety is bound to the surface.

11. The method of example 7, wherein the target region is physically separated after enzymatically destroying the non-targeted nucleic acid material.

12. The method according to any one of examples 1-11, wherein the one or more targeted endonucleases are selected from the group consisting of ribonucleoproteins, Cas enzymes, Cas9-like enzymes, Cpf1 enzymes, meganucleases , Transcription activator-like effector-based nucleases (TALENs), zinc finger nucleases, arginine nucleases, or the group consisting of combinations thereof.

13. The method of any one of examples 1-12, wherein the one or more targeted endonucleases comprise Cas9 or CPF1 or a derivative thereof.

14. The method according to any one of examples 1-13, wherein cutting the nucleic acid material comprises cutting the nucleic acid material with one or more targeted endonucleases such that more than one substantially known length is formed. target nucleic acid fragment.

15. The method of example 14, further comprising separating more than one target nucleic acid fragment based on a predetermined length.

16. The method of example 15, wherein the target nucleic acid fragments are of different substantially known lengths.

17. The method of example 15, wherein the target nucleic acid fragments each comprise related genomic sequences from one or more distinct locations in the genome.

18. The method of example 15, wherein the target nucleic acid fragments each comprise a targeted sequence from a substantially known region in the nucleic acid material.

19. The method of any one of examples 15-18, wherein separating target nucleic acid fragments based on substantially known lengths comprises by gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, filtration, or SPRI Bead purification to enrich target nucleic acid fragments.

20. The method of example 1, further comprising ligating at least one SMI and/or adaptor sequence to at least one of the 5' or 3' end of the cleaved target region of predetermined length.

21. The method of example 1, wherein the analysis comprises quantification and/or sequencing of the target region.

22. The method of example 21, wherein quantification comprises at least one of spectrophotometric analysis, real-time PCR, and/or fluorescence-based quantification.

23. The method of example 21, wherein sequencing comprises double-stranded sequencing, SPLiT-double-stranded sequencing, Sanger sequencing, shotgun sequencing, bridging amplification/sequencing, nanopore sequencing, single-molecule real-time sequencing, ion torrent sequencing, pyrosequencing Phospho-sequencing, digital sequencing (eg, digital barcode-based sequencing), direct digital sequencing, sequencing by ligation, polymerase cloning-based sequencing, current-based sequencing (eg, tunneling current), sequencing by mass spectrometry, microfluidics-based and any combination of them.

24. The method of example 21, wherein sequencing comprises:

sequencing the first strand of the target region to generate a first strand sequence read;

sequencing the second strand of the target region to generate a second strand sequence read; and

The first strand sequence reads are compared to the second strand sequence reads to generate error-corrected sequence reads.

25. The method of example 24, wherein the error-corrected sequence reads comprise nucleotide bases that are identical between the first-strand sequence reads and the second-strand sequence reads.

26. The method of example 24 or example 25, wherein variants occurring at specific positions in the error-corrected sequence reads are identified as true variants.

27. The method of any one of examples 24-26, wherein variations occurring only at specific positions in one of the first strand sequence reads or the second strand sequence reads are identified as potential artifacts.

28. The method according to any one of examples 24-27, wherein the error-corrected sequence reads are used to identify or characterize cancer, cancer risk, cancer in the organism or subject from which the double-stranded target nucleic acid molecule is derived Mutation, Cancer Metabolic Status, Mutant Phenotype, Carcinogen Exposure, Toxin Exposure, Chronic Inflammation Exposure, Age, Neurodegenerative Diseases, Pathogens, Drug Resistant Variants, Fetal Molecules, Forensic Relevant Molecules, Immunological Relevant Molecules, Mutated T cell receptors, mutated B cell receptors, mutated immunoglobulin loci, kategis loci in the genome, hypervariable loci in the genome, low frequency variants, subclonal variants, minority molecular populations, sources of contamination, Nucleic acid synthesis errors, enzyme modification errors, chemical modification errors, gene editing errors, gene therapy errors, nucleic acid information storage fragments, microbial quasispecies, virus quasispecies, organ transplantation, organ transplant rejection, cancer recurrence, residual cancer after treatment, pre-tumor A state, a dysplastic state, a microchimeric state, a stem cell transplant state, a cell therapy state, a nucleic acid marker attached to another molecule, or a combination thereof.

29. The method of any one of examples 24-27, wherein error-corrected sequence reads are used to identify mutagenic compounds or exposures.

30. The method of any one of examples 24-27, wherein error-corrected sequence reads are used to identify oncogenic compounds or exposures.

31. The method of any one of examples 24-27, wherein the nucleic acid material is from a forensic sample, and wherein the error-corrected sequence reads are used for forensic analysis.

32. The method of example 1, wherein the targeted endonucleases comprise CRISPR-associated (Cas) enzymes, ribonucleoprotein complexes, homing endonucleases, zinc finger nucleases, transcription activators At least one of a nuclease-like effector nuclease (TALEN), an arginine nuclease and/or a megaTAL nuclease.

33. The method of example 32, wherein the CRISPR-associated (Cas) enzyme is Cas9 or Cpf1.

34. The method of example 32, wherein the CRISPR-associated (Cas) enzyme is Cpfl, and wherein the target region comprises 5' overhangs and 3' overhangs of predetermined or known nucleotide sequences.

35. The method of example 1, wherein cleaving the nucleic acid material with a targeted endonuclease comprises cleaving the nucleic acid material with more than one targeted endonuclease.

36. The method of example 35, wherein the more than one targeted endonuclease comprises more than one Cas enzyme for more than one target region.

37. The method according to example 35, wherein cutting the nucleic acid material with a targeted endonuclease such that the target region of a predetermined length is separated from the rest of the nucleic acid material comprises cutting the target region with a pair of targeted endonucleases, The targeted endonuclease is directed to cleave the nucleic acid material by a predetermined distance so as to generate a target region having a predetermined length.

38. The method of example 37, wherein the pair of target endonucleases comprises a pair of Cas enzymes.

39. The method of example 38, wherein the pair of Cas enzymes comprise the same type of Cas enzyme.

40. The method of example 38, wherein the pair of Cas enzymes comprises two different types of Cas enzymes.

41. A method for enriching target nucleic acid material, comprising:

provide nucleic acid material;

cleaving the nucleic acid material with one or more targeted endonucleases such that a target region of predetermined length is separated from the remainder of the nucleic acid material, wherein the at least one targeted endonuclease includes a capture label;

capturing a target region of a predetermined length with an extraction moiety configured to bind a capture label;

release a target region of predetermined length from the targeted endonuclease; and

The cleaved target region is analyzed.

42. A method for enriching target nucleic acid material, comprising:

provide nucleic acid material;

binding catalytically inactive CRISPR-associated (Cas) enzymes to target regions of nucleic acid material;

Enzymatic treatment of nucleic acid material with one or more nucleic acid-digesting enzymes such that non-targeted nucleic acid material is destroyed and target regions are protected from the digestive enzymes by bound catalytically inactive Cas enzymes;

Release the target region from catalytically inactive Cas enzymes; and

Analyze target regions.

43. The method of example 42, wherein the binding step comprises binding a pair of catalytically inactive Cas enzymes to the target region such that nucleic acid material between the bound Cas enzymes is enzymatically protected from digestive enzymes, thereby The target nucleic acid material is enriched in the target region.

44. The method of example 42, wherein the catalytically inactive Cas enzyme comprises a capture label, and wherein the method further comprises capturing the target region with an extraction moiety configured to bind the capture label.

45. The method of example 42, further comprising enriching the target region by size selection.

46. A method for enriching target nucleic acid material, comprising:

provide nucleic acid material;

A pair of catalytically active targeted endonucleases and at least one catalytically inactive targeted endonuclease comprising a capture label are provided, wherein the catalytically inactive targeted endonuclease is directed to binding to a target region of a nucleic acid material, and wherein the pair of catalytically active targeted endonucleases are oriented to bind to the target region on either side of the catalytically inactive targeted endonuclease;

cleaving the nucleic acid material with the pair of catalytically active targeted endonucleases such that the target region is separated from the remainder of the nucleic acid material;

capturing the target region with an extraction moiety configured to bind the capture label;

release the target region from the targeted endonuclease; and

The cleaved target region is analyzed.

47. A method for enriching target nucleic acid material from a sample comprising a plurality of nucleic acid fragments, comprising:

providing one or more catalytically inactive CRISPR-associated (Cas) enzymes with capture labels to a sample comprising target nucleic acid fragments and non-target nucleic acid fragments, wherein the one or more catalytically inactive Cas enzymes are configured to bind to target nucleic acid fragment;

providing a surface comprising an extraction moiety configured to bind a capture label; and

The target nucleic acid fragments are separated from the non-target nucleic acid fragments by capturing the target nucleic acid fragments via binding of the capture label by the extraction moiety.

48. The method of example 47, further comprising ligating adaptor molecules to the ends of the plurality of nucleic acid fragments prior to providing the one or more catalytically inactive CRISPR-associated (Cas) enzymes.

49. A method for enriching target double-stranded nucleic acid material, comprising:

provide nucleic acid material;

Cleavage of nucleic acid material with one or more targeted endonucleases to generate double-stranded target nucleic acid fragments comprising 5' cohesive ends having a 5' predetermined nucleotide sequence and/or having 3' the 3' sticky ends of the predetermined nucleotide sequence; and

The double-stranded target nucleic acid molecule is separated from the remainder of the nucleic acid material by at least one of the 5' sticky ends and the 3' sticky ends.

50. The method of example 49, further comprising providing at least one sequencing adaptor molecule comprising ligatable ends that are at least partially complementary to a 5' predetermined nucleotide sequence or a 3' predetermined nucleotide sequence;

ligating at least one sequencing adaptor molecule to the double-stranded target nucleic acid molecule; and

Double-stranded target nucleic acid fragments are analyzed by sequencing.

51. The method of example 50, wherein the at least one adaptor molecule comprises a Y-shape or a U-shape.

52. The method of example 50, wherein the at least one adaptor molecule is a hairpin molecule.

53. The method of example 50, wherein the at least one adaptor molecule comprises a capture molecule configured to be bound by an extraction moiety.

54. The method of example 50, wherein the sequencing adapter molecule is ligated to each of the 5' cohesive end and the 3' cohesive end of the double-stranded target nucleic acid fragment.

55. The method of example 49, wherein separating the double-stranded target nucleic acid molecule from the rest of the nucleic acid material by at least one of the 5' cohesive end and the 3' cohesive end comprises providing an oligonucleotide, the oligonucleotide The acid has a sequence that is at least partially complementary to the 5' predetermined nucleotide sequence or the 3' predetermined nucleotide sequence.

56. The method of example 55, wherein the oligonucleotide is bound to a surface.

57. The method of example 55, wherein the oligonucleotide comprises a capture label configured to bind the extraction moiety.

58. The method of example 49, wherein the one or more targeted endonucleases comprise Cpfl.

59. The method of example 49, wherein the one or more targeted endonucleases comprise a Cas9 nickase.

60. A kit for enriching target nucleic acid material, comprising:

A nucleic acid library comprising:

nucleic acid material; and

A variety of catalytically inactive Cas enzymes, wherein the Cas enzymes include tags with sequence codes,

wherein the plurality of Cas enzymes bind to a plurality of site-specific target regions along the nucleic acid material;

Multiple probes, where each probe includes:

the oligonucleotide sequence of the complement including the corresponding sequence code; and

capture tags; and

A look-up table that classifies the relationship between site-specific target regions, sequence codes associated with the site-specific target regions, and probes that include complements of the corresponding sequence codes.

61. The method of any one of the preceding examples, wherein the nucleic acid material is or comprises at least one of double-stranded DNA and double-stranded RNA.

62. The method of any one of the preceding examples, wherein at least some of the nucleic acid material is destroyed.

63. The method of example 62, wherein the damage is or comprises oxidation, alkylation, deamination, methylation, hydrolysis, hydroxylation, nicking, intrachain crosslinking, interchain crosslinking, blunt end chain scission, Staggered-end double-strand breaks, phosphorylation, dephosphorylation, ubiquitination, glycosylation, deglycosylation, putrescylation, carboxylation, halogenation, formylation, single-strand gap, heat-induced damage, damage by drying, damage by UV exposure, damage by gamma radiation, damage by X-radiation, damage by ionizing radiation, damage by non-ionizing radiation, damage by heavy particle radiation damage, damage by nuclear decay, damage by beta radiation, damage by alpha radiation, damage by neutron radiation, damage by proton radiation, damage by cosmic radiation, damage by high pH damage, damage caused by low pH, damage caused by reactive oxidizing species, damage caused by free radicals, damage caused by peroxides, damage caused by hypochlorite, damage caused by, for example, formalin or formaldehyde, etc. damage caused by tissue fixation, damage caused by active iron, damage caused by low ionic conditions, damage caused by high ionic conditions, damage caused by unbuffered conditions, damage caused by nucleases, damage caused by environmental exposure damage, damage by fire, damage by mechanical stress, damage by enzymatic degradation, damage by microorganisms, damage by preparative mechanical shearing, damage by preparative enzymatic cleavage, natural in vivo Damages that occur, Damages that occur during nucleic acid extraction, Damages that occur during sequencing library preparation, Damages introduced by polymerases, Damages introduced during nucleic acid repair, Damages that occur during nucleic acid end tailing, During nucleic acid ligation Damage that occurs, Damage that occurs during sequencing, Damage that occurs due to mechanical processing of DNA, Damage that occurs during passage through a nanopore, Damage that occurs as part of aging in an organism, Damage that occurs due to chemical exposure of an individual damage due to mutagens, damage due to carcinogens, damage due to cleavage agents, damage due to in vivo inflammatory damage due to oxygen exposure, damage due to one or more strand breaks damage and at least one of any combination thereof.

64. The method of any one of the preceding examples, wherein the nucleic acid material is provided from a sample comprising one or more double-stranded nucleic acid molecules derived from a subject or organism.

65. The method according to example 64, wherein the sample is or comprises body tissue, biopsy sample, skin sample, blood, serum, plasma, sweat, saliva, cerebrospinal fluid, mucus, uterine lavage, vaginal swab, Pap smear Sheets, nasal swabs, oral swabs, tissue scrapings, hair, fingerprints, urine, feces, vitreous humor, peritoneal lavage, sputum, bronchial lavage, oral lavage, pleural lavage, gastric lavage Washing fluid, gastric fluid, bile, pancreatic duct lavage, bile duct lavage, common bile duct lavage, gallbladder fluid, synovial fluid, infected wounds, uninfected wounds, archaeological samples, forensic samples, water samples, tissue samples , food samples, bioreactor samples, plant samples, bacterial samples, protozoa samples, fungal samples, animal samples, virus samples, multi-organism samples, nail scrapings, semen, prostatic fluid, vaginal fluid, vaginal swabs, tubal irrigation Washes, cell-free nucleic acids, intracellular nucleic acids, metagenomic samples, lavages or swabs of implanted foreign bodies, nasal lavages, intestinal fluids, epithelial brushes, epithelial lavages, tissue biopsy samples, autopsy Samples, necropsy samples, organ samples, human identification samples, non-human identification samples, artificially generated nucleic acid samples, synthetic genetic samples, banked or stored samples, tumor tissue, fetal samples, organ transplant samples, microbial culture samples, Nuclear DNA samples, mitochondrial DNA samples, chloroplast DNA samples, acroplast DNA samples, organelle samples, and any combination thereof.

66. The method of any one of the preceding examples, wherein the nucleic acid material comprises nucleic acid molecules of substantially uniform length or near uniform length.

67. The method of any one of the preceding examples, wherein the target nucleic acid material is derived from a subject or organism.

68. The method of any one of the preceding examples, wherein the target nucleic acid material has been at least partially synthesized.

69. The method of any one of the preceding examples, wherein up to 1000 ng of nucleic acid material is initially provided.

70. The method of any one of the preceding examples, wherein at most 10 ng of nucleic acid material is initially provided.

71. The method of any one of the preceding examples, wherein the nucleic acid material comprises nucleic acid material from more than one source.

Equivalents and Scope

The foregoing detailed description of embodiments of the present technology is not intended to be exhaustive or to limit the present technology to the precise forms disclosed above. While specific embodiments of, and examples for, the technology have been described above for illustrative purposes, various equivalent modifications are possible within the scope of the technology, as those skilled in the relevant art will recognize. For example, although the steps are presented in a given order, alternative embodiments may perform the steps in a different order. The various embodiments described herein can also be combined to provide further embodiments. All references cited herein are incorporated by reference as if fully set forth herein.

From the foregoing, it will be appreciated that specific embodiments of the technology have been described herein for illustrative purposes, but well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of embodiments of the technology . Singular or plural terms may also include plural or singular terms, respectively, where the context allows. Furthermore, while advantages associated with certain embodiments of the technology have been described in the context of those embodiments, other embodiments may exhibit such advantages, and not all embodiments need to exhibit such advantages in order to fall within the scope of this technology. Accordingly, the present disclosure and related technology may encompass other embodiments not expressly shown or described herein.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosed technology described herein. The scope of the present technology is not intended to be limited by the above description, but rather is as set forth in the appended claims.

Claims (60)

1. A method for enriching a target nucleic acid material, comprising:

providing a nucleic acid material;

Cleaving the nucleic acid material with one or more targeted endonucleases such that a target region of a predetermined length is separated from the remainder of the nucleic acid material;

enzymatically disrupting non-targeted nucleic acid material;

releasing the target region of the predetermined length from the targeted endonuclease; and

the cleaved target region is analyzed.

2. The method of claim 1, wherein enzymatically disrupting non-targeted nucleic acid material comprises providing an exonuclease.

3. The method of claim 1, wherein enzymatically disrupting non-targeted nucleic acid material comprises providing one or more of an exonuclease and an endonuclease.

4. The method of claim 1, wherein disrupting comprises at least one of enzymatic digestion and enzymatic cleavage.

5. The method of any one of claims 1-4, wherein the one or more targeted endonucleases remain bound to the target region during the enzymatic disruption step.

6. The method according to any one of claims 1-5, wherein at least one targeted endonuclease is a ribonucleoprotein complex comprising a capture label, and wherein the target region of predetermined length is physically separated from the remainder of the nucleic acid by the capture label while the at least one targeted endonuclease remains bound to the target region.

7. The method of any one of claims 1-5, wherein at least one targeted endonuclease is a ribonucleoprotein complex comprising a capture label, and wherein the method further comprises capturing the target region with an extraction moiety configured to bind the capture label.

8. The method of claim 6 or claim 7, wherein the capture label is or comprises at least one of: acridine, azide (NHS ester), digitoxin (NHS ester), I-linker, amino modifier C6, amino modifier C12, amino modifier C6 dT, Unilink amino modifier, hexynyl, 5-octadiynyl dU, biotin (azide), biotin dT, biotin TEG, bis-biotin, PC biotin, desthiobiotin TEG, thiol modifier C3, dithiol, thiol modifier C6S-S, succinyl group.

9. The method of claim 7, wherein the extraction moiety is or comprises at least one of an aminosilane, an epoxysilane, an isothiocyanate, an aminophenylsilane, an aminopropylsilane, a mercaptosilane, an aldehyde, an epoxide, a phosphonate, streptavidin, avidin, a hapten for a recognition antibody, a specific nucleic acid sequence, a magnetically attractable particle (Dynabeads), and a photolabile resin.

10. The method of claim 7, wherein the extraction moiety is bound to a surface.

11. The method of claim 7, wherein the target region is physically separated after enzymatically disrupting the non-targeted nucleic acid material.

12. The method of any one of claims 1-11, wherein the one or more targeted endonucleases are selected from the group consisting of ribonucleoproteins, Cas enzymes, Cas 9-like enzymes, Cpf1 enzymes, meganucleases, transcription activator-like effector based nucleases (TALENs), zinc finger nucleases, arginine nucleases, or combinations thereof.

13. The method of any one of claims 1-12, wherein the one or more targeted endonucleases comprise Cas9 or CPF1 or derivatives thereof.

14. The method of any one of claims 1-13, wherein cleaving the nucleic acid material comprises cleaving the nucleic acid material with one or more targeted endonucleases such that more than one target nucleic acid fragment of substantially known length is formed.

15. The method of claim 14, further comprising isolating the more than one target nucleic acid fragments based on the predetermined length.

16. The method of claim 15, wherein the target nucleic acid fragments have different substantially known lengths.

17. The method of claim 15, wherein the target nucleic acid fragments each comprise related genomic sequences from one or more different locations in a genome.

18. The method of claim 15, wherein the target nucleic acid fragments each comprise a targeted sequence from a substantially known region within the nucleic acid material.

19. The method of any one of claims 15-18, wherein isolating the target nucleic acid fragments based on a substantially known length comprises enriching the target nucleic acid fragments by gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, filtration, or SPRI bead purification.

20. The method of claim 1, further comprising ligating at least one SMI and/or adaptor sequence to at least one of the 5 'or 3' ends of a predetermined length of the cleaved target region.

21. The method of claim 1, wherein analyzing comprises quantification and/or sequencing of the target region.

22. The method of claim 21, wherein quantifying comprises at least one of spectrophotometric analysis, real-time PCR, and/or fluorescence-based quantification.

23. The method of claim 21, wherein sequencing comprises double-stranded sequencing, SPLiT-double-stranded sequencing, Sanger sequencing, shotgun sequencing, bridge amplification/sequencing, nanopore sequencing, single molecule real-time sequencing, ion torrent sequencing, pyrosequencing, digital sequencing (e.g., digital barcode-based sequencing), direct digital sequencing, sequencing by ligation, polymerase clone-based sequencing, current-based sequencing (e.g., tunneling current), sequencing by mass spectrometry, microfluidic-based sequencing, and any combination thereof.

24. The method of claim 21, wherein sequencing comprises:

sequencing a first strand of the target region to generate first strand sequence reads;

sequencing a second strand of the target region to generate second strand sequence reads; and

comparing the first strand sequence reads to the second strand sequence reads to generate error-corrected sequence reads.

25. The method of claim 24, wherein the error-corrected sequence reads comprise nucleotide bases that are identical between the first strand sequence reads and the second strand sequence reads.

26. The method of claim 24 or claim 25, wherein a variation occurring at a particular position in the error-corrected sequence reads is identified as a true variant.

27. The method of any one of claims 24-26, wherein variations that occur only at specific positions in one of the first strand sequence reads or the second strand sequence reads are identified as potential artifacts.

28. The method of any one of claims 24-27, wherein the error-corrected sequence reads are used to identify or characterize cancer, cancer risk, cancer mutation, cancer metabolic state, mutation phenotype, carcinogen exposure, toxin exposure, chronic inflammatory exposure, age, neurodegenerative disease, pathogen, drug-resistant variant, fetal molecule, forensic-related molecule, immunologically-related molecule, mutated T cell receptor, mutated B cell receptor, mutated immunoglobulin locus, kategis site in genome, hypervariable site in genome, low frequency variant, subcloned variant, minority molecule population, contamination source, nucleic acid synthesis error, enzymatic modification error, chemical modification error, gene editing error, gene therapy error, nucleic acid information storage fragment, nucleic acid in an organism or subject from which a double-stranded target nucleic acid molecule is derived, A microbial quasispecies, a viral quasispecies, an organ transplant rejection, a cancer recurrence, a post-treatment residual cancer, a pre-neoplastic state, a dysplastic state, a micro-chimeric state, a stem cell transplant state, a cell therapy state, a nucleic acid marker attached to another molecule, or a combination thereof.

29. The method of any one of claims 24-27, wherein the error corrected sequence reads are used to identify mutagenic compounds or exposures.

30. The method of any one of claims 24-27, wherein the error corrected sequence reads are used to identify an oncogenic compound or exposure.

31. The method of any one of claims 24-27, wherein the nucleic acid material is from a forensic sample, and wherein the error corrected sequence reads are used for forensic analysis.

32. The method of claim 1, wherein the targeted endonuclease comprises at least one of a CRISPR-associated (Cas) enzyme, a ribonucleoprotein complex, a homing endonuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an arginine nuclease, and/or a megaTAL nuclease.

33. The method of claim 32, wherein the CRISPR-associated (Cas) enzyme is Cas9 or Cpf 1.

34. The method of claim 32, wherein the CRISPR-associated (Cas) enzyme is Cpf1, and wherein the target region comprises a 5 'overhang and a 3' overhang of a predetermined or known nucleotide sequence.

35. The method of claim 1, wherein cleaving the nucleic acid material with a targeted endonuclease comprises cleaving the nucleic acid material with more than one targeted endonuclease.

36. The method of claim 35, wherein the more than one targeted endonuclease comprises more than one Cas enzyme for more than one target region.

37. The method of claim 35, wherein cleaving the nucleic acid material with a targeted endonuclease such that a target region of a predetermined length is separated from the remainder of the nucleic acid material comprises cleaving the target region with a pair of targeted endonucleases, the targeted endonucleases being oriented to cleave the nucleic acid material at a predetermined distance so as to generate the target region of a predetermined length.

38. The method of claim 37, wherein the pair of target nucleic acid endonucleases comprises a pair of Cas enzymes.

39. The method of claim 38, wherein the pair of Cas enzymes comprises the same type of Cas enzyme.

40. The method of claim 38, wherein the pair of Cas enzymes comprises two different types of Cas enzymes.

41. A method for enriching a target nucleic acid material, comprising:

providing a nucleic acid material;

cleaving the nucleic acid material with one or more targeted endonucleases such that a target region of a predetermined length is separated from the remainder of the nucleic acid material, wherein at least one targeted endonuclease includes a capture label;

Capturing the target region of the predetermined length with an extraction moiety configured to bind the capture label;

releasing the target region of the predetermined length from the targeted endonuclease; and

the cleaved target region is analyzed.

42. A method for enriching a target nucleic acid material, comprising:

providing a nucleic acid material;

binding a catalytically inactive CRISPR-associated (Cas) enzyme to a target region of the nucleic acid material;

enzymatically treating the nucleic acid material with one or more nucleic acid digesting enzymes such that non-targeted nucleic acid material is destroyed and the target region is protected from the digesting enzymes by a bound catalytically inactive Cas enzyme;

releasing the target region from the catalytically inactive Cas enzyme; and

analyzing the target region.

43. The method of claim 42, wherein the binding step comprises binding a pair of catalytically inactive Cas enzymes to the target region such that nucleic acid material between the bound Cas enzymes is enzymatically protected from the digestive enzymes, thereby enriching the target nucleic acid material of the target region.

44. The method of claim 42, wherein the catalytically inactive Cas enzyme comprises a capture label, and wherein the method further comprises capturing the target region with an extraction moiety configured to bind the capture label.

45. The method of claim 42, further comprising enriching the target region by size selection.

46. A method for enriching a target nucleic acid material, comprising:

providing a nucleic acid material;

providing a pair of catalytically active targeted endonucleases and at least one catalytically inactive targeted endonuclease comprising capture tags, wherein the catalytically inactive targeted endonuclease is oriented to bind to the target region of the nucleic acid material, and wherein the pair of catalytically active targeted endonucleases are oriented to bind to the target region on either side of the catalytically inactive targeted endonuclease;

cleaving the nucleic acid material with the pair of catalytically active targeted endonucleases such that the target region is separated from the remainder of the nucleic acid material;

capturing the target region with an extraction moiety configured to bind the capture label;

releasing the target region from the targeted endonuclease; and

the cleaved target region is analyzed.

47. A method for enriching a target nucleic acid material from a sample comprising a plurality of nucleic acid fragments, comprising:

providing one or more catalytically inactive CRISPR-associated (Cas) enzymes with a capture label to a sample comprising a target nucleic acid fragment and a non-target nucleic acid fragment, wherein the one or more catalytically inactive Cas enzymes are configured to bind to the target nucleic acid fragment;

Providing a surface comprising an extraction moiety configured to bind to the capture label; and

separating the target nucleic acid fragments from the non-target nucleic acid fragments by capturing the target nucleic acid fragments via binding of the capture label by the extraction portion.

48. The method of claim 47, further comprising ligating an adaptor molecule to the ends of the plurality of nucleic acid fragments prior to providing the one or more catalytically inactive CRISPR-associated (Cas) enzymes.

49. A method for enriching a target double-stranded nucleic acid material, comprising:

providing a nucleic acid material;

cleaving the nucleic acid material with one or more targeted endonucleases to generate double-stranded target nucleic acid fragments comprising a 5 'sticky end having a 5' predetermined nucleotide sequence and/or a 3 'sticky end having a 3' predetermined nucleotide sequence; and

separating the double stranded target nucleic acid molecule from the remainder of the nucleic acid material by at least one of the 5 'sticky end and the 3' sticky end.

50. The method of claim 49, further comprising providing at least one sequencing adaptor molecule comprising an ligatable end at least partially complementary to said 5 'predetermined nucleotide sequence or said 3' predetermined nucleotide sequence;

Ligating said at least one sequencing adaptor molecule to said double stranded target nucleic acid molecule; and

analyzing the double-stranded target nucleic acid fragments by sequencing.

51. The method of claim 50, wherein the at least one adaptor molecule comprises a Y-shape or a U-shape.

52. The method of claim 50, wherein the at least one adaptor molecule is a hairpin molecule.

53. The method of claim 50, wherein the at least one adaptor molecule comprises a capture molecule configured to be bound by an extraction moiety.

54. The method of claim 50, wherein a sequencing adaptor molecule is ligated to each of the 5 'sticky end and the 3' sticky end of the double-stranded target nucleic acid fragments.

55. The method of claim 49, wherein separating the double stranded target nucleic acid molecule from the remainder of the nucleic acid material by at least one of the 5 'sticky end and the 3' sticky end comprises providing an oligonucleotide having a sequence at least partially complementary to the 5 'predetermined nucleotide sequence or the 3' predetermined nucleotide sequence.

56. The method of claim 55, wherein the oligonucleotide is bound to a surface.

57. The method of claim 55, wherein the oligonucleotide comprises a capture label configured to bind to an extraction moiety.

58. The method of claim 49, wherein the one or more targeted endonucleases comprise Cpf 1.

59. The method of claim 49, wherein the one or more targeted endonucleases comprise a Cas9 nickase.

60. A kit for enriching a target nucleic acid material, comprising:

a nucleic acid library comprising:

a nucleic acid material; and

a plurality of catalytically inactive Cas enzymes, wherein the Cas enzymes comprise a tag having a sequence code,

wherein the plurality of Cas enzymes are bound to a plurality of site-specific target regions along the nucleic acid material;

a plurality of probes, wherein each probe comprises:

a complement oligonucleotide sequence comprising a corresponding sequence code; and

capturing the tag; and

a look-up table that classifies relationships between the site-specific target region, sequence codes associated with the site-specific target region, and probes of a complement including the corresponding sequence codes.

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